PATATIN-LIKE PHOSPHOLIPASE DOMAIN CONTAINING 3 (PNPLA3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting the Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene, and methods of using such RNAi agents to inhibit expression of a PNPLA3 gene and methods of treating subjects having Nonalcoholic Fatty Liver Disease (NAFLD) and/or a PNPLA3-associated disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/267,615, filed on Feb. 5, 2019, which is a continuation of U.S. patent application Ser. No. 15/670,132, filed on Aug. 7, 2017 now U.S. Pat. No. 10,231,988 issued on Mar. 19, 2019, which is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2016/017550, filed on Feb. 11, 2016, to U.S. Provisional Patent Application No. 62/115,724, filed on Feb. 13, 2015, and to U.S. Provisional Patent Application No. 62/266,818, filed on Dec. 14, 2015. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 12, 2021, is named 121301_03205_SL.txt and is 529,923 bytes in size.

BACKGROUND OF THE INVENTION

The accumulation of excess triglyceride in the liver is known as hepatic steatosis (or fatty liver), and is associated with adverse metabolic consequences, including insulin resistance and dyslipidemia. Fatty liver is frequently found in subjects having excessive alcohol intake and subjects having obesity, diabetes, or hyperlipidemia. However, in the absence of excessive alcohol intake (>10 g/day), nonalcoholic fatty liver disease (NAFLD) can develop. NAFLD refers to a wide spectrum of liver diseases that can progress from simple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). All of the stages of NAFLD have in common the accumulation of fat (fatty infiltration) in the liver cells (hepatocytes).

The NAFLD spectrum begins with and progress from its simplest stage, called simple fatty liver (steatosis). Simple fatty liver involves the accumulation of fat (triglyceride) in the liver cells with no inflammation (hepatitis) or scarring (fibrosis). The next stage and degree of severity in the NAFLD spectrum is NASH, which involves the accumulation of fat in the liver cells, as well as inflammation of the liver. The inflammatory cells destroy liver cells (hepatocellular necrosis), and NASH ultimately leads to scarring of the liver (fibrosis), followed by irreversible, advanced scarring (cirrhosis). Cirrhosis that is caused by NASH is the last and most severe stage in the NAFLD spectrum.

In 2008, a genomewide association study of individuals with proton magnetic resonance spectroscopy of the liver to evaluate hepatic fat content, a significant association was identified between hepatic fat content and the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) gene (see, for example, Romeo et al. (2008) Nat. Genet., 40(12):1461-1465). Studies with knock-in mice have demonstrated that expression of a sequence polymorphism (rs738409, I148M) in PNPLA3 causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of triglycerides in the liver (Smagris et al. (2015) Hepatology, 61:108-118). Specifically, the PNPLA3 I148M variant was associated with promoting the development of fibrogenesis by activating the hedgehog (Hh) signaling pathway, leading to the activation and proliferation of hepatic stellate cells and excessive generation and deposition of extracellular matrix (Chen et al. (2015) World J. Gastroenterol., 21(3):794-802).

Currently, treatments for NAFLD are directed towards weight loss and treatment of any secondary conditions, such as insulin resistance or dyslipidemia. To date, no pharmacologic treatments for NAFLD have been approved. Therefore, there is a need for therapies for subjects suffering from NAFLD.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PNPLA3 gene. The PNPLA3 gene may be within a cell, e.g., a cell within a subject, such as a human.

In one aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of any of the sequences in any one of Tables 3-5, 7, and 8.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3-5, 7, and 8.

In one embodiment, the double stranded RNAi agent comprises at least one modified nucleotide. In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic. In another embodiment, the modified nucleotides comprise a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In one embodiment, the region of complementarity is at least 17 nucleotides in length. In another embodiment, the region of complementarity is between 19 and 21 nucleotides in length. In another embodiment, the region of complementarity is 19 nucleotides in length. In another embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In one embodiment, the double stranded RNAi agent further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. In another embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In one embodiment the ligand is

In another embodiment, the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic:

and, wherein X is O or S. In one embodiment, the X is O.

In one embodiment, the region of complementarity comprises one of the antisense sequences in any one of Tables 3-5, 7, and 8. In another embodiment, the region of complementarity consists of one of the antisense sequences in any one of Tables 3-5, 7, and 8.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented b formula III):

(III) sense: 5′ n_(p)-N_(a)-(X X X)_(i)-Nb-Y Y Y-N_(b)-(Z Z Z)_(l)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)k-N_(b)′-Y′Y′Y′-N_(b)′- (Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; 1 is 0; k is 1; 1 is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end. In one embodiment, the Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′.

In another embodiment, formula (III) is represented by formula (IIIb):

(IIIb) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′- N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIIc):

(IIIc) sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b)-Y′Y′Y′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIId):

(IIId) sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

In one embodiment, the double stranded region is 15-30 nucleotide pairs in length. In another embodiment, the double stranded region is 17-23 nucleotide pairs in length. In another embodiment, the double stranded region is 17-25 nucleotide pairs in length. In another embodiment, the double stranded region is 23-27 nucleotide pairs in length. In another embodiment, the double stranded region is 19-21 nucleotide pairs in length. In another embodiment, the double stranded region is 21-23 nucleotide pairs in length.

In one embodiment, each strand has 15-30 nucleotides. In another embodiment, each strand has 19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In one embodiment, the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sense strand.

In one embodiment, the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic

In one embodiment, the double stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In one embodiment, he strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In another embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. In another embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification. In another embodiment, the Y′ nucleotides contain a 2′-O-methyl modification. In another embodiment, p′>0. In another embodiment, p′=2. In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In another embodiment, all n_(p)′ are linked to neighboring nucleotides via phosphorothioate linkages.

In one embodiment, the double stranded RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3-5, 7, and 8. In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III)  sense: 5′ n_(p)-N_(a)-(X X X)_(i)-Nb-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′- (Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′

wherein i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not be present independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein: i, j, k, and l are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

In another embodiment, the invention provides a double stranded-ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein i, j, k, and l are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III)  sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein i, j, k, and l are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(IIIa)  sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′

wherein each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In another aspect, the invention provides a double stranded-ribonucleic acid (RNAi) agent for inhibiting expression of PNPLA3, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In another embodiment, each strand has 19-30 nucleotides.

In another aspect, the invention provides a cell containing the double stranded RNAi agent as described herein.

In another aspect, the invention provides a vector encoding at least one strand of a double stranded RNAi agent, wherein the double stranded RNAi agent comprises a region of complementarity to at least a part of an mRNA encoding PNPLA3, wherein the double stranded RNAi agent is 30 base pairs or less in length, and wherein the double stranded RNAi agent targets the mRNA for cleavage. In one embodiment, the region of complementarity is at least 15 nucleotides in length. In another embodiment, the region of complementarity is 19 to 21 nucleotides in length.

In another aspect, the invention provides a cell comprising a vector as described herein.

In another aspect, the invention provides a pharmaceutical composition for inhibiting expression of a PNPLA3 gene comprising the double stranded RNAi agent of the invention. In one embodiment, the double stranded RNAi agent is administered in an unbuffered solution. In another embodiment, the unbuffered solution is saline or water. In another embodiment, the double stranded RNAi agent is administered with a buffer solution. In another embodiment, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In another embodiment, the buffer solution is phosphate buffered saline (PBS).

In another aspect, the invention provides a pharmaceutical composition comprising the double stranded RNAi agent of the invention and a lipid formulation. In one embodiment, the lipid formulation comprises a LNP. In another embodiment, the lipid formulation comprises a MC3.

In another aspect, the invention provides a method of inhibiting PNPLA3 expression in a cell, the method comprising (a) contacting the cell with the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the cell. In one embodiment, the cell is within a subject. In another embodiment, the subject is a human. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the PNPLA3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

In another aspect, the invention provides a method of treating a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In another aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a prophylactically effective amount of the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 expression.

In one embodiment, the administration of the double stranded RNAi to the subject causes a decrease in the hedgehog signaling pathway.

In one embodiment, the PNPLA3-associated disease is a PNPLA3-associated disease. In another embodiment, the PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is fatty liver (steatosis). In another embodiment, the PNPLA3-associated disease is nonalcoholic steatohepatitis (NASH). In another embodiment, the PNPLA3-associated disease is obesity. In one embodiment, the subject is human. In another embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the subject has a PNPLA3 I148M mutation. In one embodiment, the mutation is heterozygous. In another embodiment, the mutation is homozygous.

In another embodiment, the invention further comprises administering an anti-PNPLA3 antibody, or antigen-binding fragment thereof, to the subject.

In one embodiment, the double stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In one embodiment, the dsRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In another embodiment, the dsRNA agent is administered at a dose selected from the group consisting of 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, the double stranded RNAi agent is administered to the subject once a week. In another embodiment, the double stranded RNAi agent is administered to the subject once a month.

In one embodiment, the double stranded RNAi agent is administered to the subject subcutaneously.

In another embodiment, the methods of the invention further comprise measuring hedgehog signaling pathway levels in the subject. In one embodiment, a decrease in the levels of expression or activity of the hedgehog (Hh) signaling pathway indicate that the PNPLA3-associated disease is being treated or prevented.

The present invention is further illustrated by the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percentage of PNPLA3 mRNA remaining in the liver of ob/ob mice following administration of a single dose of 0.3 mg/kg, 1.5 mg/kg, or 3.0 mg/kg of the indicated iRNA agents.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (PNPLA3 gene) in mammals.

The RNAi agents of the invention have been designed to target the human PNPLA3 gene, including portions of the gene that are conserved in the PNPLA3 orthologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites and/or the specific modifications in these RNAi agents confer to the RNAi agents of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention also provides methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a PNPLA3 gene, e.g., an PNPLA3-associated disease, such as Nonalcoholic Fatty Liver Disease (NAFLD), using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PNPLA3 gene.

Very low dosages of the iRNAs of the invention, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of the corresponding gene (PNPLA3 gene).

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a PNPLA3 gene.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an angiotensinogen gene as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of a PNPLA3 gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, “Patatin-Like Phospholipase Domain Containing 3,” used interchangeably with the term “PNPLA3,” refers to the naturally occurring gene that encodes a triacylglycerol lipase that mediates triacyl glycerol hydrolysis in adipocytes. The amino acid and complete coding sequences of the reference sequence of the human PNPLA3 gene may be found in, for example, GenBank Accession No. GI:17196625 (RefSeq Accession No. NM_025225.2; SEQ ID NO:1; SEQ ID NO:2). Mammalian orthologs of the human PNPLA3 gene may be found in, for example, GenBank Accession Nos. GI: 544461323 (RefSeq Accession No. XM_005567051.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8); GI: 544461325 (RefSeq Accession No. XM_005567052.1, cynomolgus monkey; SEQ ID NO:11 and SEQ ID NO:12); GI:297261270 (RefSeq Accession No. XM_001109144.2, rhesus monkey, SEQ ID NO:9 and SEQ ID NO:10); GI:144226244 (RefSeq Accession No. NM_054088.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:537361027 (RefSeq Accession No. NM_001282324.1, rat; SEQ ID NO:5 and SEQ ID NO:6).

Additional examples of PNPLA3 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an PNPLA3 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an PNPLA3 gene. In one embodiment, the target sequence is within the protein coding region of PNPLA3.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of a PNPLA3 gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a PNPLA3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an PNPLA3 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., an PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAi agent is a dsRNA that is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a PNPLA3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an PNPLA3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5′- and/or 3′-terminus of the iRNA. In one embodiment, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the antisense strand. In another embodiment, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3′-terminus of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a PNPLA3 gene). For example, a polynucleotide is complementary to at least a part of an PNPLA3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a PNPLA3 gene.

Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target PNPLA3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:2, or, or a fragment of SEQ ID NO:2, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In another embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of the sense strands in any one of Tables 3-5, 7, and 8, or a fragment of any one of the sense strands in any one of Tables 3-5, 7, and 8, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression and/or replication; a human at risk for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression; a human having a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression, as described herein. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with PNPLA3 gene expression and/or PNPLA3 protein production, e.g., the presence of increased protein activity in the hedgehog (Hh) signaling pathway, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of PNPLA3 gene expression and/or PNPLA3 protein production in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more and is preferably down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an PNPLA3 gene and/or production of PNPLA3 protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of PNPLA3 gene expression, such as the presence of elevated levels of proteins in the hedgehog signaling pathway, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “Patatin-Like Phospholipase Domain Containing 3-associated disease” or “PNPLA3-associated disease,” is a disease or disorder that is caused by, or associated with PNPLA3 gene expression or PNPLA3 protein production. The term “PNPLA3-associated disease” includes a disease, disorder or condition that would benefit from a decrease in PNPLA3 gene expression, replication, or protein activity. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic steatohepatitis (NASH). In another embodiment, the PNPLA3-associated disease is liver cirrhosis. In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance. In one embodiment, the PNPLA3-associated disease is obesity.

In one embodiment, an PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). As used herein, “nonalcoholic fatty liver disease,” used interchangeably with the term “NAFLD,” refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol ingestion per day. NAFLD is the most common liver disease in the United States, and is commonly associated with insulin resistance/type 2 diabetes mellitus and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which are incorporated herein by reference.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a patient for treating a subject having PNPLA3-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by PNPLA3 gene expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject who does not yet experience or display symptoms of a PNPLA3-associated disease, but who may be predisposed, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. RNAi agents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes), the retina or parts of the retina (e.g., retinal pigment epithelium), the central nervous system or parts of the central nervous system (e.g., ventricles or choroid plexus), or the pancreas or certain cells or parts of the pancreas. In some embodiments, a “sample derived from a subject” refers tocerebrospinal fluid obtained from the subject. In preferred embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject. In further embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of a PNPLA3 gene. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a PNPLA3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having an PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD). The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an PNPLA3 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the PNPLA3 gene, the iRNA inhibits the expression of the PNPLA3 gene (e.g., a human, a primate, a non-primate, or a bird PNPLA3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an PNPLA3 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target PNPLA3 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3-5, 7, and 8, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 3-5, 7, and 8. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an PNPLA3 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 3-5, 7, and 8, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 3-5, 7, and 8. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in any one of Tables 3, 4, and 7 are not described as modified and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3-5, 7, and 8, or the sequences of any one of Tables 3-5, 7, and 8 that are modified, or the sequences of any one of Tables 3-5, 7, and 8 that are conjugated. In other words, the invention encompasses dsRNA of any one of Tables 3-5, 7, and 8 which are unmodified, un-conjugated, modified, and/or conjugated, as described herein.

In another aspect, a double stranded ribonucleic acid (dsRNA) of the invention for inhibiting expression of PNPLA3 comprises, consists essentially of, or consists of a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of a sense strand in any one of Tables 3-5, 7, and 8 and the antisense strand comprises the nucleotide sequence of the corresponding antisense strand in any one of Tables 3-5, 7, and 8.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Table 3-5, 7, and 8, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of any one of Tables 3-5, 7, and 8 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of any one of Tables 3-5, 7, and 8, and differing in their ability to inhibit the expression of a PNPLA3 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 3-5, 7, and 8 identify a site(s) in a PNPLA3 transcript that is susceptible to RISC-mediated cleavage (see, e.g., Table 9). As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in any one of Tables 3-5, 7, and 8 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a PNPLA3 gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. Thus, while the sequences identified, for example, in any one of Tables 3-5, 7, and 8 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in any one of Tables 3-5, 7, and 8, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an PNPLA3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an PNPLA3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an PNPLA3 gene is important, especially if the particular region of complementarity in an PNPLA3 gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is unmodified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified iRNAs of the invention in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(−n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,283); 4′-CH2 N(OCH3)-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. patents and US patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and 3-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. A “hydroxymethyl substituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, also referred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, the entire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or PCT Application No. PCT/US2012/065691, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The RNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand. The resulting RNAi agents present superior gene silencing activity.

More specifically, it has been surprisingly discovered that when the sense strand and antisense strand of the double stranded RNAi agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of an RNAi agent, the gene silencing activity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., PNPLA3 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may range from 12-30 nucleotides in length. For example, each strand may be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhang regions and/or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3-terminal end of the sense strand or, alternatively, at the 3-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^(st) nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^(st) paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ” “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In one embodiment, the RNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′-3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′-3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.

In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif For example, the portion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_(a)” and “N_(b)” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_(a) and N_(b) can be the same or different modifications. Alternatively, N_(a) and/or N_(b) may be present or absent when there is a wing modification present.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and/or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-terminus or the 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

(I) 5′ n_(p)-N_(a)-(X X X )_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(Ib) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Id) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6 Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′. 

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

(II) 5′ n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(i)-N′_(a)-n_(p)′ 3′

wherein:

-   -   k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

(Ib) 5′ n_(q′)-N_(a′)-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p′) 3′; (Ic) 5′ n_(q′)-N_(a′)-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′)-3′; or (Id) 5′ n_(q′)-N_(a′)-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a′)-n_(p′) 3′. 

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula:

(Ia) 5′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 3′. 

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(l)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb) 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIc) 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)-X′X′X′-N_(b)-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIId)  5′ n_(p)-N_(a)-XXX-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-X′X′X′-N_(b)-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′ >0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 3-5, 7, and 8. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 14) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 15) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 16) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include HBV and above (e.g., HBV, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, disorder or condition associated with PNPLA3 gene expression) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention iRNA targeting the PNPLA3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an PNPLA3 gene.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular, (IM), or intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of an PNPLA3 gene.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PNPLA3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months). After an initial treatment regimen, the treatments can be administered on a less frequent basis.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as a disorder that would benefit from reduction in the expression of PNPLA3. Such models can be used for in vivo testing of an agent, as well as for determining a therapeutically effective dose. Suitable dietary and genetic mouse models are reviewed in Kanuri and Bergheim (Int. J. Mol. Sci. (2013) 14:11963-11980).

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate iRNA. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M)i, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M)i or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNA agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implementations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA agent can be delivered, for example, subcutaneously by infection in order to deliver iRNA agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs, of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle. The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP- 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG- 1 dimethylaminopropane cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC 2,2-Dilinoleyl-4- XTC/DPPC/Cholesterol/PEG-cDMA dimethylaminoethyl- 57.1/7.1/34.4/1.4 [1,3]-dioxolane (XTC) lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 57.5/7.5/31.5/3.5 [1,3]-dioxolane (XTC) lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 57.5/7.5/31.5/3.5 [1,3]-dioxolane (XTC) lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 60/7.5/31/1.5, [1,3]-dioxolane (XTC) lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 60/7.5/31/1.5, [1,3]-dioxolane (XTC) lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 50/10/38.5/1.5 [1,3]-dioxolane (XTC) Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N- ALN100/DSPC/Cholesterol/ dimethyl-2,2- PEG-DMG di((9Z,12Z)-octadeca- 50/10/38.5/1.5 9,12-dienyl)tetrahydro- Lipid:siRNA 10:1 3aH-cyclo- penta[d][1,3]dioxol- 5-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)- MC-3/DSPC/Cholesterol/ heptatriaconta- PEG-DMG 6,9,28,31-tetraen- 50/10/38.5/1.5 19-yl 4-(dimethyl- Lipid:siRNA 10:1 amino)butanoate (MC3) LNP12 1,1′-(2-(4-(2-((2- Tech G1/DSPC/Cholesterol/ (bis(2-hydroxy- PEG-DMG dodecyl)amino)- 50/10/38.5/1.5 ethyl)(2-hydroxy Lipid:siRNA 10:1 dodecyl)-amino)- ethyl)piperazin- 1-yl)ethylazanediyl)- didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/ GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. W02009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference. XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference. MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference. ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference. C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a) D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating a hemolytic disorder. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent.

In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PNPLA3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Methods For Inhibiting PNPLA3 Expression

The present invention also provides methods of inhibiting expression of a PNPLA3 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of PNPLA3 in the cell, thereby inhibiting expression of PNPLA3 in the cell.

Contacting of a cell with an RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any other ligand that directs the RNAi agent to a site of interest.

In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a PNPLA3” is intended to refer to inhibition of expression of any PNPLA3 gene (such as, e.g., a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene) as well as variants or mutants of a PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (such as a mutant PNPLA3 gene giving rise to amyloid deposition), or a transgenic PNPLA3 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a PNPLA3 gene” includes any level of inhibition of a PNPLA3 gene, e.g., at least partial suppression of the expression of a PNPLA3 gene. The expression of the PNPLA3 gene may be assessed based on the level, or the change in the level, of any variable associated with PNPLA3 gene expression, e.g., PNPLA3 mRNA level, PNPLA3 protein level, or the number or extent of amyloid deposits. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with PNPLA3 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of a PNPLA3 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Inhibition of the expression of a PNPLA3 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a PNPLA3 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of a PNPLA3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a PNPLA3 gene may be assessed in terms of a reduction of a parameter that is functionally linked to PNPLA3 gene expression, e.g., PNPLA3 protein expression or Hedgehog pathway protein activities. PNPLA3 gene silencing may be determined in any cell expressing PNPLA3, either constitutively or by genomic engineering, and by any assay known in the art.

Inhibition of the expression of a PNPLA3 protein may be manifested by a reduction in the level of the PNPLA3 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of a PNPLA3 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of PNPLA3 mRNA that is expressed by a cell or group of cells, or the level of circulating PNPLA3 mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of PNPLA3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the PNPLA3 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating PNPLA3 mRNA may be detected using methods the described in PCT/US2012/043584, the entire contents of which are hereby incorporated herein by reference.

In one embodiment, the level of expression of PNPLA3 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific PNPLA3. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of PNPLA3 mRNA.

An alternative method for determining the level of expression of PNPLA3 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of PNPLA3 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

The expression levels of PNPLA3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of PNPLA3 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

The level of PNPLA3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in a symptom of a PNPLA3 disease, such as reduction in edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; or abdominal pain. These symptoms may be assessed in vitro or in vivo using any method known in the art.

In some embodiments of the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of PNPLA3 may be assessed using measurements of the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells that express a particular type of receptor.

VIII. Methods of Treating or Preventing PNPLA3-Associated Diseases

The present invention provides therapeutic and prophylactic methods which include administering to a subject with a PNPLA3-associated disease, disorder, and/or condition, or prone to developing, a PNPLA3-associated disease, disorder, and/or condition, compositions comprising an iRNA agent, or pharmaceutical compositions comprising an iRNA agent, or vectors comprising an iRNA of the invention. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In one embodiment, the PNPLA3-associated disease is NAFLD. In another embodiment, the PNPLA3-associated disease is NASH. In another embodiment, the PNPLA3-associated disease is fatty liver (steatosis). In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance.

The methods of the invention are useful for treating a subject having a PNPLA3-associated disease, e.g., a subject that would benefit from reduction in PNPLA3 gene expression and/or PNPLA3 protein production. In one aspect, the present invention provides methods of reducing the level of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene expression in a subject having nonalcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods of reducing the level of PNPLA3 protein in a subject with NAFLD. The present invention also provides methods of reducing the level of activity of the hedgehog pathway in a subject with NAFLD.

In another aspect, the present invention provides methods of treating a subject having an NAFLD. In one aspect, the present invention provides methods of treating a subject having an PNPLA3-associated disease, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of an iRNA agent of the invention targeting a PNPLA3 gene or a pharmaceutical composition comprising an iRNA agent of the invention targeting a PNPLA3 gene or a vector of the invention comprising an iRNA agent targeting an PNPLA3 gene.

In one aspect, the invention provides methods of preventing at least one symptom in a subject having NAFLD, e.g., the presence of elevated hedgehog signaling pathways, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider-like blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid build up and swelling of the legs (edema), abdomen swelling (ascites), and mental confusion. The methods include administering to the subject a therapeutically effective amount of the iRNA agent, e.g. dsRNA, pharmaceutical compositions, or vectors of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 gene expression.

In another aspect, the present invention provides uses of a therapeutically effective amount of an iRNA agent of the invention for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression.

In a further aspect, the present invention provides uses of an iRNA agent, e.g., a dsRNA, of the invention targeting an PNPLA3 gene or pharmaceutical composition comprising an iRNA agent targeting an PNPLA3 gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production, such as a subject having a disorder that would benefit from reduction in PNPLA3 gene expression, e.g., a PNPLA3-associated disease.

In another aspect, the invention provides uses of an iRNA, e.g., a dsRNA, of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production.

In a further aspect, the present invention provides uses of an iRNA agent of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or SCAP protein production, such as a PNPLA3-associated disease.

In one embodiment, an iRNA agent targeting PNPLA3 is administered to a subject having a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD), such that the expression of a PNPLA3 gene, e.g., in a cell, tissue, blood or other tissue or fluid of the subject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more when the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering a composition described herein such that expression of the target PNPLA3 gene is decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target PNPLA3 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

Administration of the dsRNA according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD). By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 4%5% 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of NAFLD may be assessed, for example, by periodic monitoring of NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting PNPLA3 or pharmaceutical composition thereof, “effective against” an PNPLA3-associated disease indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating NAFLD and/or an PNPLA3-associated disease and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. In one embodiment, subjects can be administered 0.5 mg/kg of the dsRNA. Values and ranges intermediate to the recited values are also intended to be part of this invention.

Administration of the iRNA can reduce the presence of PNPLA3 protein levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on PNPLA3 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where the modified or unmodified iRNA agent is directly suspended in aqueous or suitable buffer solvent, as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The free iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of PNPLA3 gene expression are those having nonalcoholic fatty liver disease (NAFLD) and/or an PNPLA3-associated disease or disorder as described herein.

Treatment of a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression includes therapeutic and prophylactic treatment.

The invention further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of PNPLA3 gene expression, e.g., a subject having a PNPLA3-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

For example, in certain embodiments, an iRNA targeting a PNPLA3 gene is administered in combination with, e.g., an agent useful in treating an PNPLA3-associated disease as described elsewhere herein. For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in PNPLA3 expression, e.g., a subject having a PNPLA3-associated disease, include an iRNA agent targeting a different portion of the PNPLA3 gene, a therapeutic agent, and/or procedures for treating a PNPLA3-associated disease or a combination of any of the foregoing.

In certain embodiments, a first iRNA agent targeting a PNPLA3 gene is administered in combination with a second iRNA agent targeting a different portion of the PNPLA3 gene. For example, the first RNAi agent comprises a first sense strand and a first antisense strand forming a double stranded region, wherein substantially all of the nucleotides of said first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, wherein said first sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker; and the second RNAi agent comprises a second sense strand and a second antisense strand forming a double stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, all of the nucleotides of the first and second sense strand and/or all of the nucleotides of the first and second antisense strand comprise a modification.

In one embodiment, the at least one of the modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic.

In certain embodiments, a first iRNA agent targeting a PNPLA3 gene is administered in combination with a second iRNA agent targeting a gene that is different from the PNPLA3 gene. For example, the iRNA agent targeting the PNPLA3 gene may be administered in combination with an iRNA agent targeting the SCAP gene. The first iRNA agent targeting a PNPLA3 gene and the second iRNA agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of the same pharmaceutical composition. Alternatively, the first iRNA agent targeting a PNPLA3 gene and the second iRNA agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of different pharmaceutical compositions.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

The present invention also provides methods of using an iRNA agent of the invention and/or a composition containing an iRNA agent of the invention to reduce and/or inhibit PNPLA3 expression in a cell. In other aspects, the present invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting PNPLA3 gene expression in a cell. In yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 gene expression in a cell are provided. In still other aspects, the present invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting PNPLA3 protein production in a cell. In yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 protein production in a cell are provided. The methods and uses include contacting the cell with an iRNA, e.g., a dsRNA, of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene or inhibiting PNPLA3 protein production in the cell.

Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of PNPLA3 may be determined by determining the mRNA expression level of PNPLA3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of PNPLA3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, flow cytometry methods, ELISA, and/or by determining a biological activity of PNPLA3.

In the methods and uses of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an PNPLA3 gene, e.g., a cell from a subject having NAFLD or a cell comprising an expression vector comprising a PNPLA3 gene or portion of a PNPLA3 gene. A cell suitable for use in the methods and uses of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell.

PNPLA3 gene expression may be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

PNPLA3 protein production may be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PNPLA3 gene of the mammal to be treated. When the organism to be treated is a human, the composition can be administered by any means known in the art including, but not limited to subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. In one embodiment, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of PNPLA3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the subject.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an PNPLA3 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal for use in inhibiting expression of the PNPLA3 gene in the mammal. In another aspect, the present invention provides use of an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the PNPLA3 gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human, a composition comprising an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sample of the iRNA-administered subject by any methods known it the art, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g., ELISA or Western blotting, described herein. In one embodiment, a tissue sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target in vivo following administration of iRNA agent is done by performing 5′-RACE or modifications of the protocol as known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al. (2006) Nature 441: 111-4).

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

siRNA Design

A set of iRNAs targeting human PNPLA3, “Patatin-Like Phospholipase Domain Containing 3” (RefSeq Accession No. NM_025225, GI:17196625; SEQ ID NO:1 and SEQ ID NO:2) and PNPLA3 orthologs from toxicology species (for example, GenBank Accession Nos. GI: 544461323 (REFSEQ Accession No. XM_005567051.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8); GI: 544461325 (RefSeq Accession No. XM_005567052.1, cynomolgus monkey; SEQ ID NO:11 and SEQ ID NO:12); GI:297261270 (RefSeq Accession No. XM_001109144.2, rhesus monkey, SEQ ID NO:9 and SEQ ID NO:10); GI:144226244 (RefSeq Accession No. NM_054088.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:537361027 (RefSeq Accession No. NM_001282324.1, rat; SEQ ID NO:5 and SEQ ID NO: 6)) were designed using custom R and Python scripts.

The human PNPLA3 RefSeq mRNA has a length of 2805 bases. The rationale and method for the set of iRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 1 through position 2805 of human PNPLA3 mRNA (containing the coding region) was determined using a linear model that predicted the direct measure of mRNA knockdown based on the data of more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weights for the mismatches were 2.8 for seed mismatches, 1.2 for cleavage site mismatches, and 1 for mismatches in other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was greater than or equal to 3.0 and predicted efficacy was greater than or equal to 70% knockdown of the PNPLA3 transcript. One set of iRNAs containing structure-activity modifications, including various 2′-O-methyl and 2′-fluoro substitution patterns, were also designed, synthesized and screened.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 3.

siRNA Synthesis

PNPLA3 iRNA sequences were synthesized at 1 μmol scale on a Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support is controlled pore glass (500 A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids), 5′phosphate and other modifications were introduced using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 minutes employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagents at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that were protected with a tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection was performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 uL of dimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent were added and the solution was incubated for additional 20 minutes at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and was precipitated by addition of 1 mL of acetontile:ethanol mixture (9:1). The plates were cooled at −80 C for 2 hours, supernatant was decanted carefully with the aid of a multi channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and was desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity.

Annealing of PNPLA3 single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96-well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 μM in 1×PBS.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine -3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96 N[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinol Hyp-(GalNAc-alkyl)3 (dt) 2′-deoxythymidine-3′-phosphate Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy- 3-phosphate (abasic 2′-OMe furanose) Y44 2-hydroxymethyl-tetrahydrofurane-5-phosphate (Agn) Adenosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer (Cgn) Cytidine-glycol nucleic acid (GNA) P Phosphate VP Vinyl-phosphate

TABLE 3 Unmodified Sense and Antisense Strand Sequences of PNPLA3 RNAi agents  Sense SEQ Anti- Target Se- sense SEQ Site in Start quence ID Sequence  ID Other  Gen Bank Oligo Name Position (5′-3′) NO (5′-3′) NO Set Ref. No. NM_025225.2_219- 217 GGCUU 19 UUGGU 111 hcmr 217-239 240_C21_Asense CCUGG AGAAG GCUUC CCCAG UACCA GAAGC A CGC NM_054088.3_250- 248 UAUAA 20 UCCAU 112 mr 248-270 271_sense UGGAG GAGGA AUCCU UCUCC CAUGG AUUAU A ACG NM_025225.2_388- 386 UUGUG 21 UACUC 113 hc 386-408 409_E21A_sense CGGAA CUGGC GGCCA CUUCC GGAGU GCACA A AGA NM_025225.2_396- 394 AAGGC 22 AAUGU 114 hc 394-416 417_sense CAGGA UCCGA GUCGG CUCCU AACAU GGCCU U UCC NM_025225.2_397- 395 AGGCC 23 UAAUG 115 hc 395-417 418_G21_Asenseense AGGAG UUCCG UCGGA ACUCC ACAUU UGGCC A UUC NM_054088.3_443_- 441 GUGUC 24 UUUGG 116 mr 441-463 464_senseense UGAGU AAUGG UCCAU AACUC UCCAA AGACA A CCA NM_054088.3_469- 467 AGUCG 25 UACAC 117 mr 467-489 490_G21_Asenseense UGGAU CAGGG GCCCU CAUCC GGUGU ACGAC A UUC NM_025225.2_549- 547 AACGU 26 AAAGU 118 hc 547-569 570_sense UCUGG CAGAC UGUCU ACCAG GACUU AACGU U UUU NM_025225.2_562- 560 CUGAC 27 UGUCU 119 he 560-582 583_G21A_sense UUUCG UUGGA GUCCA CCGAA AAGAC AGUCA A GAC NM_025225.2_569- 567 UCGGU 28 ACGAC 120 hc 567-589 590_sense CCAAA UUCGU GACGA CUUUG AGUCG GACCG U AAA NM_025225.2_570- 568 CGGUC 29 UACGA 121 he 568-590 591_G21A_sense CAAAG CUUCG ACGAA UCUUU GUCGU GGACC A GAA NM_025225.2_579- 577 GACGA 30 UAAGG 122 he 577-599 600_G21_Asense AGUCG CAUCC UGGAU ACGAC GCCUU UUCGU A CUU NM_025225.2_596- 594 CUUGG 31 AUGAA 123 hemr 594-616 617_sense UAUGU GCAGG UCCUG AACAU CUUCA ACCAA U GGC NM_025225.2_630- 628 GGCCU 32 UAAGG 124 hemr 628-650 651C21_Asense UAUCC AAGGA CUCCU GGGAU UCCUU AAGGC A CAC NM_025225.2_674- 672 AGGAG 33 UGUAC 125 he 672-694 695_C21_Asense UGAGU GUUGU GACAA CACUC CGUAC ACUCC A UCC NM_025225.2_678- 676 GUGAG 34 UAAGG 126 he 676-698 699_C21A_sense UGACA GUACG ACGUA UUGUC CCCUU ACUCA A CUC NM_025225.2_701- 699 UGAUG 35 UUGAU 127 he 699-721 722_C21_Asense CCAAA GGUUG ACAAC UUUUG CAUCA GCAUC A AAU NM_025225.2_746- 744 CGACA 36 UUGAC 128 he 744-766 767_sense UCUGC UUUAG CCUAA GGCAG AGUCA AUGUC A GUA NM_054088.3_770- 768 UGCUA 37 UUCCA 129 mr 768-790 791C21_Asense UCAAG GGUAC GGUAC CCUUG CUGGA AUAGC A ACA NM_025225.2_771- 769 ACGAA 38 UUCCA 130 he 769-791 792_C21_Asense CUUUC CAUGA UUCAU AGAAA GUGGA GUUCG A UGG NM_025225.2_817- 815 GCACA 39 UAAGG 131 he 815-837 838_C21_Asense GGGAA UAGAG CCUCU GUUCC ACCUU CUGUG A CAG NM_025225.2_871- 869 UGCUG 40 UAAGG 132 he 869-891 892_C21_Asense GGAGA CAUAU GAUAU CUCUC GCCUU CCAGC A ACC NM_025225.2_874- 872 UGGGA 41 UUCGA 133 he 872-894 895_G21A_sense GAGAU AGGCA AUGCC UAUCU UUCGA CUCCC A AGC NM_025225.2_878- 876 AGAGA 42 UAUCC 134 he 876-898 899_sense UAUGC UCGAA CUUCG GGCAU AGGAU AUCUC A UCC NM_025225.2_882- 880 AUAUG 43 UAAAU 135 he 880-902 903_G21A_sense CCUUC AUCCU GAGGA CGAAG UAUUU GCAUA A UCU NM_025225.2_885- 883 UGCCU 44 AUCCA 136 he 883-905 906_sense UCGAG AAUAU GAUAU CCUCG UUGGA AAGGC U AUA NM_025225.2_908- 906 AUUCA 45 UUCUC 137 he 906-928 929_sense GGUUC UUCCA UUGGA AGAAC AGAGA CUGAA A UGC NM_025225.2_964- 962 CAUCC 46 UAUCC 138 he 962-984 985_C21A_sense UCAGA AUCCC AGGGA UUCUG UGGAU AGGAU A GAC NM_025225.2_1100- 1098 CCUGC 47 AUGCU 139 he 1098-1120 1121_sense CCUGG CUCAU GAUGA CCCAG GAGCA GGCAG U GAU NM_054088.3_1163- 1161 UCCCA 48 UAUUC 140 mr 1161-1183 1184_G21A_sense GGUUU GGGCA GUGCC CAAAC CGAAU CUGGG A AUG NM_054088.3_1165- 1163 CCAGG 49 UUCAU 141 mr 1163-1185 1186_C21_Asense UUUGU UCGGG GCCCG CACAA AAUGA ACCUG A GGA NM_025225.2_1173- 1171 GACAA 50 UCUCA 142 he 1171-1193 1194_C21_Asense AGGUG UGUAU GAUAC CCACC AUGAG UUUGU A CUU NM_025225.2_1176- 1174 AAAGG 51 UUUGC 143 he 1174-1196 1197_G21_Asense UGGAU UCAUG ACAUG UAUCC AGCAA ACCUU A UGU NM_025225.2_1180- 1178 GUGGA 52 AAAUC 144 he 1178-1200 1201_se_use UACAU UUGCU GAGCA CAUGU AGAUU AUCCA U CCU NM_025225.2_1181- 1179 UGGAU 53 UAAAU 145 he 1179-1201 1202_G21_Asense ACAUG CUUGC AGCAA UCAUG GAUUU UAUCC A ACC NM_025225.2_1184- 1182 AUACA 54 UUGCA 146 he 1182-1204 1205_sense UGAGC AAUCU AAGAU UGCUC UUGCA AUGUA A UCC NM_025225.2_1191- 1189 AGCAA 55 UAGCA 147 he 1189-1211 1212_sense GAUUU AGUUG GCAAC CAAAU UUGCU CUUGC A UCA NM_025225.2_1193- 1191 CAAGA 56 UGUAG 148 he 1191-1213 1214C21_Asense UUUGC CAAGU AACUU UGCAA GCUAC AUCUU A GCU NM_025225.2_1196- 1194 GAUUU 57 AUGGG 149 he 1194-1216 1217_sense GCAAC UAGCA UUGCU AGUUG ACCCA CAAAU U CUU NM_025225.2_1200- 1198 UGCAA 58 UCUAA 150 he 1198-1220 1221_G21A_sense CUUGC UGGGU UACCC AGCAA AUUAG GUUGC A AAA NM_025225.2_1203- 1201 AACUU 59 UAUCC 151 he 1201-1223 1224_sense GCUAC UAAUG CCAUU GGUAG AGGAU CAAGU A UGC NM_025225.2_1266- 1264 GCCAU 60 UCUCU 152 he 1264-1286 1287_sense UGCGA GGACA UUGUC AUCGC CAGAG AAUGG A CAG NM_025225.2_1274- 1272 GAUUG 61 UUCAC 153 he 1272-1294 1295_C21_A_sense UCCAG CAGUC AGACU UCUGG GGUGA ACAAU A CGC NM_025225.2_1288- 1286 UGGUG 62 UAUCU 154 he 1286-1308 1309_sense ACAUG GGAAG GCUUC CCAUG CAGAU UCACC A AGU NM_025225.2_1302- 1300 CCAGA 63 UACAU 155 he 1300-1322 1323_C21A_sense UAUGC CGUCG CCGAC GGCAU GAUGU AUCUG A GAA NM_025225.2_1325- 1323 GUGGU 64 UAGGU 156 he 1323-1345 1346_C21_Asense UGCAG CACCC UGGGU ACUGC GACCU AACCA A CAG NM_025225.2_1389- 1387 AGGUC 65 UCUCA 157 he 1387-1409 1410_C21A_sense CCAAA CUGGC UGCCA AUUUG GUGAG GGACC A UGG NM_025225.2_1621- 1619 UCACU 66 UAGAC 158 he 1619-1641 1642_sense UGAGG UCGCC AGGCG UCCUC AGUCU AAGUG A ACU NM_025225.2__1636- 1634 AGUCU 67 UCUGA 159 he 1634-1656 1657_sense AGCAG AAGAA AUUCU UCUGC UUCAG UAGAC A UCG NM_025225.2_1646- 1644 AUUCU 68 UUUUA 160 he 1644-1666 1667_G21_Asense UUCAG GCACC AGGUG UCUGA CUAAA AAGAA A UCU NM_025225.2_1647- 1645 UUCUU 69 ACUUU 161 he 1645-1667 1668_sense UCAGA AGCAC GGUGC CUCUG UAAAG AAAGA U AUC NM_025225.2__1658- 1656 GUGCU 70 AAAGA 162 he 1656-1678 1679_sense AAAGU UGGGA UUCCC AACUU AUCUU UAGCA U CCU NM_025225.2_1669- 1667 UCCCA 71 UGUAG 163 he 1667-1689 1690_C21A_sense UCUUU CUGCA GUGCA CAAAG GCUAC AUGGG A AAA NM_025225.2_1713- 1711 CUGCC 72 UAUCC 164 he 1711-1733 1734_C21A_sense UGUGA UCCAC CGUGG GUCAC AGGAU AGGCA A GGG NM_025225.2_1718- 1716 UGUGA 73 UCUGG 165 he 1716-1738 1739_C21_A_sense CGUGG GAUCC AGGAU UCCAC CCCAG GUCAC A AGG NM_025225.2_1740- 1738 UCUGA 74 AUAAA 166 he 1738-1760 1761_sense GCUGA ACCAA GUUGG CUCAG UUUUA CUCAG U AGG NM_025225.2__1741_- 1739 CUGAG 75 UAUAA 167 he 1739-1761 1762_G21_A_sense CUGAG AACCA UUGGU ACUCA UUUAU GCUCA A GAG NM_025225.2_1749- 1747 AGUUG 76 UAGCU 168 he 1747-1769 1770_sense GUUUU UUUCA AUGAA UAAAA AAGCU CCAAC A UCA NM_025225.2_1751- 1749 UUGGU 77 UCUAG 169 he 1749-1771 1772_G21_Asense UUUAU CUUUU GAAAA CAUAA GCUAG AACCA A ACU NM_025225.2_1753- 1751 GGUUU 78 UUCCU 170 he 1751-1773 1774_sense UAUGA AGCUU AAAGC UUCAU UAGGA AAAAC A CAA NM_025225.2_1754- 1752 GUUUU 79 UUUCC 171 he 1752-1774 1775_G21_Asense AUGAA UAGCU AAGCU UUUCA AGGAA UAAAA A CCA NM_025225.2_1755- 1753 UUUUA 80 UCUUC 172 he 1753-1775 1776_C21_Asense UGAAA CUAGC AGCUA UUUUC GGAAG AUAAA A ACC NM_025225.2_1758- 1756 UAUGA 81 UUUGC 173 he 1756-1778 1779_C21_Asense AAAGC UUCCU UAGGA AGCUU AGCAA UUCAU A AAA NM_025225.2_1827- 1825 CGUUA 82 UCCCA 174 he 1825-1847 1848_sense AUUCA ACCAG GCUGG CUGAA UUGGG UUAAC A GCA NM_025225.2_1828- 1826 GUUAA 83 UUCCC 175 he 1826-1848 1849_sense UUCAG AACCA CUGGU GCUGA UGGGA AUUAA A CGC NM_025225.2_1836- 1834 AGCUG 84 UGUGU 176 he 1834-1856 1857_C21A_sense GUUGG CAUUU GAAAU CCCAA GACAC CCAGC A UGA NM_025225.2_1900- 1898 CCUAU 85 AACAG 177 he 1898-1920 1921_sense UAAUG UCUGA GUCAG CCAUU ACUGU AAUAG U GGC NM_025225.2_1901_- 1899 CUAUU 86 UAACA 178 he 1899-1921 1922_C21A_sense AAUGG GUCUG UCAGA ACCAU CUGUU UAAUA A GGG NM_025225.2_1984- 1982 GCUGG 87 UAAGA 179 he 1982-2004 2005_G21_Asense CCCAU UCACA GUGUG CAUGG AUCUU GCCAG A CCU NM_025225.2_1986- 1984 UGGCC 88 UACAA 180 he 1984-2006 2007_G21_Asense CAUGU GAUCA GUGAU CACAU CUUGU GGGCC A AGC NM_025225.2_2190- 2188 CCUAA 89 UUAAA 181 he 2188-2210 2211_sense CUAAA CAUUA AUAAU UUUUA GUUUA GUUAG A GUG NM_025225.2_2243- 2241 UUACC 90 AAUAC 182 he 2241-2263 2264_sense UGUUG AAAAU AAUUU UCAAC UGUAU AGGUA U ACA NM_025225.2_2245- 2243 ACCUG 91 AUAAU 183 he 2243-2265 2266_sense UUGAA ACAAA UUUUG AUUCA UAUUA ACAGG U UAA NM_025225.2_2258- 2256 UGUAU 92 UUCAC 184 he 2256-2278 2279_G21_Asense UAUGU UGAUU GAAUC CACAU AGUGA AAUAC A AAA NM_025225.2_2263- 2261 UAUGU 93 AACAU 185 he 2261-2283 2284_sense GAAUC CUCAC AGUGA UGAUU GAUGU CACAU U AAU NM_025225.2_2278- 2276 GAUGU 94 AAGGC 186 he 2276-2298 2299_sense UAGUA UUAUU GAAUA CUACU AGCCU AACAU U CUC NM_025225.2_2279- 2277 AUGUU 95 UAAGG 187 he 2277-2299 2300_sense AGUAG CUUAU AAUAA UCUAC GCCUU UAACA A UCU NM_054088.3_3032- 3030 UGGAG 96 UAUCU 188 mr 3030-3052 3053_G21A_sense CAACA AGACA GUGUC CUGUU UAGAU GCUCC A AGA NM_054088.3_3106- 3104 CUUUU 97 UUUCC 189 mr 3104-3126 3127_G21_Asense GGAGG UAGCU CAGCU GCCUC AGGAA CAAAA A GUA NM_054088.3_3226- 3224 AAGAC 98 UAAAC 190 mr 3224-3246 3247_sense AAUGA ACCAA UUUGG AUCAU UGUUU UGUCU A UUG NM_054088.3_3228- 3226 GACAA 99 UCUAA 191 mr 3226-3248 3249_sense UGAUU ACACC UGGUG AAAUC UUUAG AUUGU A CUU NM_054088.3_3230- 3228 CAAUG 100 UUUCU 192 mr 3228-3250 3251_sense AUUUG AAACA GUGUU CCAAA UAGAA UCAUU A GUC NM_054088.3_3447- 3445 UGCCA 101 AAAGU 193 mr 3445-3467 3468_sense GAUAA AAUAA CUUAU GUUAU UACUU CUGGC U AGG NM_054088.3_3473- 3471 ACACC 102 AUUAG 194 mr 3471-3493 3494_sense UUUGG UAAGA CUCUU GCCAA ACUAA AGGUG U UCC NM_054088.3_3629- 3627 CUGGC 103 UAUAC 195 mr 3627-3649 3650_sense UCCAA AAAGA AUCUU UUUGG UGUAU AGCCA A GUG NM_054088.3_3630- 3628 UGGCU 104 UUAUA 196 mr 3628-3650 3651_G21A_sense CCAAA CAAAG UCUUU AUUUG GUAUA GAGCC A AGU NM_054088.3_3635- 3633 CCAAA 105 UAUGA 197 mr 3633-3655 3656_C21_Asense UCUUU CUAUA GUAUA CAAAG GUCAU AUUUG A GAG NM_054088.3_3986- 3984 AGAGA 106 UAGCC 198 mr 39844006 4007_sense CAAAG UAGAC UGUCU ACUUU AGGCU GUCUC A UAG NM_054088.3_3993- 3991 AAGUG 107 UUCUG 199 mr 3991-4013 4014_sense UCUAG UGUAG GCUAC CCUAG ACAGA ACACU A UUG NM_054088.3_4283- 4281 AGAAA 108 UAAAG 200 mr 4281-4303 4304_G21_Asense CUUCU CAAGG GCCUU CAGAA GCUUU GUUUC A UAC NM_054088.3_4540- 4538 GAAGG 109 UGUGU 201 mr 4538-4560 4561_C21_Asense AUUGA AUCCA AUGGA UUCAA UACAC UCCUU A CUG NM_054088.3_4543- 4541 GGAUU 110 UUUGG 202 mr 4541-4563 4564_sense GAAUG UGUAU GAUAC CCAUU ACCAA CAAUC A CUU

Example 2. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

siRNA Design

A set of iRNAs targeting the human PNPLA3 (human: NCBI refseqID NM_025225; NCBI GeneID: 80339), as well as toxicology-species PNPLA3 orthologs (cynomolgus monkey: XM_005567051; mouse: NM_054088; rat: XM_006242109) were designed using custom R and Python scripts. The human PNPLA3 REFSEQ mRNA has a length of 2805 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 174 through position 2805 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey, mouse, and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, e.g., positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, e.g., positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8, 1.2 and 1 for seed mismatches, cleavage site, and other positions up through antisense position 19, respectively. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=3.0 and predicted efficacy was >=70% knockdown of the PNPLA3 transcript.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 4. A detailed list of the modified PNPLA3 sense and antisense strand sequences is shown in Table 5.

In Vitro Screening

Cell Culture and Transfections

Hep3b cells were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of iRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Fifty μl of EMEM containing ˜5×10³ cells were then added to the iRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 20 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905), 0.5 μl PNPLA3 probe (Hs00228747_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, or mock transfected cells. The results from the assays are shown in Table 6.

TABLE 4 Unmodified Sense and Antisense Strand Sequences of PNPLA3 RNAi Agents  Start Nucleotide Site Range Sense  Sense SEQ Antisense Antisense SEQ in in Lex Oligo Sequence  ID Oligo Sequence ID NM_ NM_ Name Name (5′-3′) NO Name (5′-3′) NO 025225.2 025225.2 AD-68792.1 A-138374.1 GGGGGCGGGGCUGACGUCA 203 A-138375.1 UGACGUCAGCCCCGCCCCC 454 11 11-29 AD-68793.1 A-138376.1 ACGUCGCGCUGGGAAUGCA 204 A-138377.1 UGCAUUCCCAGCGCGACGU 455 24 24-42 AD-68794.1 A-138378.1 GGAAUGCCCUGGCCGAGAA 205 A-138379.1 UUCUCGGCCAGGGCAUUCC 456 35 35-53 AD-68795.1 A-138380.1 UGGCCGAGACACUGAGGCA 206 A-138381.1 UGCCUCAGUGUCUCGGCCA 457 44 44-62 AD-68796.1 A-138382.1 UGAGGCAGGGUAGAGAGCA 207 A-138383.1 UGCUCUCUACCCUGCCUCA 458 56 56-74 AD-68797.1 A-138384.1 AGAGAGCGCUUGCGGGCGA 208 A-138385.1 UCGCCCGCAAGCGCUCUCU 459 67 67-85 AD-68798.1 A-138386.1 CGGGCGCCGGGCGGAGCUA 209 A-138387.1 UAGCUCCGCCCGGCGCCCG 460 79 79-97 AD-68799.1 A-138388.1 GCGGAGCUGCUGCGGAUCA 210 A-138389.1 UGAUCCGCAGCAGCUCCGC 461 89  89-107 AD-68800.1 A-138390.1 UGCGGAUCAGGACCCGAGA 211 A-138391.1 UCUCGGGUCCUGAUCCGCA 462 99  99-117 AD-68801.1 A-138392.1 ACCCGAGCCGAUUCCCGAU 212 A-138393.1 AUCGGGAAUCGGCUCGGGU 463 110 110-128 AD-68802.1 A-138394.1 UUCCCGAUCCCGACCCAGA 213 A-138395.1 UCUGGGUCGGGAUCGGGAA 464 121 121-139 AD-68803.1 A-138396.1 ACCCAGAUCCUAACCCGCA 214 A-138397.1 UGCGGGUUAGGAUCUGGGU 465 133 133-151 AD-68804.1 A-138398.1 UAACCCGCGCCCCCGCCCA 215 A-138399.1 UGGGCGGGGGCGCGGGUUA 466 143 143-161 AD-68805.1 A-138400.1 CCGCCCCGCCGCCGCCGCA 216 A-138401.1 UGCGGCGGCGGCGGGGCGG 467 155 155-173 AD-68806.1 A-138402.1 CGCCGCCAUGUACGACGCA 217 A-138403.1 UGCGUCGUACAUGGCGGCG 468 167 167-185 AD-68807.1 A-138404.1 UACGACGCAGAGCGCGGCU 218 A-138405.1 AGCCGCGCUCUGCGUCGUA 469 177 177-195 AD-68808.1 A-138406.1 CGCGGCUGGAGCUUGUCCU 219 A-138407.1 AGGACAAGCUCCAGCCGCG 470 189 189-207 AD-68809.1 A-138408.1 AGCUUGUCCUUCGCGGGCU 220 A-138409.1 AGCCCGCGAAGGACAAGCU 471 198 198-216 AD-68810.1 A-138410.1 CGCGGGCUGCGGCUUCCUA 221 A-138411.1 UAGGAAGCCGCAGCCCGCG 472 209 209-227 AD-68811.1 A-138412.1 UUCCUGGGCUUCUACCACA 222 A-138413.1 UGUGGUAGAAGCCCAGGAA 473 222 222-240 AD-68812.1 A-138414.1 UUCUACCACGUCGGGGCGA 223 A-138415.1 UCGCCCCGACGUGGUAGAA 474 231 231-249 AD-68813.1 A-138416.1 CGGGGCGACCCGCUGCCUA 224 A-138417.1 UAGGCAGCGGGUCGCCCCG 475 242 242-260 AD-68814.1 A-138418.1 UGCCUGAGCGAGCACGCCA 225 A-138419.1 UGGCGUGCUCGCUCAGGCA 476 255 255-273 AD-68815.1 A-138420.1 AGCACGCCCCGCACCUCCU 226 A-138421.1 AGGAGGUGCGGGGCGUGCU 477 265 265-283 AD-68816.1 A-138422.1 ACCUCCUCCGCGACGCGCA 227 A-138423.1 UGCGCGUCGCGGAGGAGGU 478 277 277-295 AD-68817.1 A-138424.1 GCGACGCGCGCAUGUUGUU 228 A-138425.1 AACAACAUGCGCGCGUCGC 479 286 286-304 AD-68818.1 A-138426.1 UGUUGUUCGGCGCUUCGGA 229 A-138427.1 UCCGAAGCGCCGAACAACA 480 298 298-316 AD-68819.1 A-138428.1 CUUCGGCCGGGGCGUUGCA 230 A-138429.1 UGCAACGCCCCGGCCGAAG 481 310 310-328 AD-68820.1 A-138430.1 GGCGUUGCACUGCGUCGGA 231 A-138431.1 UCCGACGCAGUGCAACGCC 482 320 320-338 AD-68821.1 A-138432.1 UGCGUCGGCGUCCUCUCCA 232 A-138433.1 UGGAGAGGACGCCGACGCA 483 330 330-348 AD-68822.1 A-138434.1 UCUCCGGUAUCCCGCUGGA 233 A-138435.1 UCCAGCGGGAUACCGGAGA 484 343 343-361 AD-68823.1 A-138436.1 UCCCGCUGGAGCAGACUCU 234 A-138437.1 AGAGUCUGCUCCAGCGGGA 485 352 352-370 AD-68824.1 A-138438.1 CAGACUCUGCAGGUCCUCU 235 A-138439.1 AGAGGACCUGCAGAGUCUG 486 363 363-381 AD-68825.1 A-138440.1 UCCUCUCAGAUCUUGUGCA 236 A-138441.1 UGCACAAGAUCUGAGAGGA 487 376 376-394 AD-68826.1 A-138442.1 UCUUGUGCGGAAGGCCAGA 237 A-138443.1 UCUGGCCUUCCGCACAAGA 488 386 386-404 AD-68827.1 A-138444.1 AAGGCCAGGAGUCGGAACA 238 A-138445.1 UGUUCCGACUCCUGGCCUU 489 396 396-414 AD-68828.1 A-138446.1 CGGAACAUUGGCAUCUUCA 239 A-138447.1 UGAAGAUGCCAAUGUUCCG 490 408 408-426 AD-68829.1 A-138448.1 GCAUCUUCCAUCCAUCCUU 240 A-138449.1 AAGGAUGGAUGGAAGAUGC 491 418 418-436 AD-68830.1 A-138450.1 CCAUCCUUCAACUUAAGCA 241 A-138451.1 UGCUUAAGUUGAAGGAUGG 492 429 429-447 AD-68831.1 A-138452.1 UUAAGCAAGUUCCUCCGAA 242 A-138453.1 UUCGGAGGAACUUGCUUAA 493 441 441-459 AD-68832.1 A-138454.1 CCUCCGACAGGGUCUCUGA 243 A-138455.1 UCAGAGACCCUGUCGGAGG 494 452 452-470 AD-68833.1 A-138456.1 UCUCUGCAAAUGCCUCCCA 244 A-138457.1 UGGGAGGCAUUUGCAGAGA 495 464 464-482 AD-68834.1 A-138458.1 UGCCUCCCGGCCAAUGUCA 245 A-138459.1 UGACAUUGGCCGGGAGGCA 496 474 474-492 AD-68835.1 A-138460.1 AAUGUCCACCAGCUCAUCU 246 A-138461.1 AGAUGAGCUGGUGGACAUU 497 486 486-504 AD-68836.1 A-138462.1 AGCUCAUCUCCGGCAAAAU 247 A-138463.1 AUUUUGCCGGAGAUGAGCU 498 496 496-514 AD-68837.1 A-138464.1 CGGCAAAAUAGGCAUCUCU 248 A-138465.1 AGAGAUGCCUAUUUUGCCG 499 506 506-524 AD-68838.1 A-138466.1 AUCUCUCUUACCAGAGUGU 249 A-138467.1 ACACUCUGGUAAGAGAGAU 500 519 519-537 AD-68839.1 A-138468.1 ACCAGAGUGUCUGAUGGGA 250 A-138469.1 UCCCAUCAGACACUCUGGU 501 528 528-546 AD-68840.1 A-138470.1 AUGGGGAAAACGUUCUGGU 251 A-138471.1 ACCAGAACGUUUUCCCCAU 502 541 541-559 AD-68841.1 A-138472.1 ACGUUCUGGUGUCUGACUU 252 A-138473.1 AAGUCAGACACCAGAACGU 503 550 550-568 AD-68842.1 A-138474.1 UCUGACUUUCGGUCCAAAG 253 A-138475.1 CUUUGGACCGAAAGUCAGA 504 561 561-579 AD-68843.1 A-138476.1 UCCAAAGACGAAGUCGUGA 254 A-138477.1 UCACGACUUCGUCUUUGGA 505 573 573-591 AD-68844.1 A-138478.1 AAGUCGUGGAUGCCUUGGU 255 A-138479.1 ACCAAGGCAUCCACGACUU 506 583 583-601 AD-68845.1 A-138480.1 CCUUGGUAUGUUCCUGCUU 256 A-138481.1 AAGCAGGAACAUACCAAGG 507 595 595-613 AD-68846.1 A-138482.1 UCCUGCUUCAUCCCCUUCU 257 A-138483.1 AGAAGGGGAUGAAGCAGGA 508 606 606-624 AD-68847.1 A-138484.1 UCCCCUUCUACAGUGGCCU 258 A-138485.1 AGGCCACUGUAGAAGGGGA 509 616 616-634 AD-68848.1 A-138486.1 AGUGGCCUUAUCCCUCCUU 259 A-138487.1 AAGGAGGGAUAAGGCCACU 510 627 627-645 AD-68849.1 A-138488.1 CCUCCUUCCUUCAGAGGCA 260 A-138489.1 UGCCUCUGAAGGAAGGAGG 511 639 639-657 AD-68850.1 A-138490.1 UCAGAGGCGUGCGAUAUGU 261 A-138491.1 ACAUAUCGCACGCCUCUGA 512 649 649-667 AD-68851.1 A-138492.1 GAUAUGUGGAUGGAGGAGU 262 A-138493.1 ACUCCUCCAUCCACAUAUC 513 661 661-679 AD-68852.1 A-138494.1 GAGGAGUGAGUGACAACGU 263 A-138495.1 ACGUUGUCACUCACUCCUC 514 673 673-691 AD-68853.1 A-138496.1 UGACAACGUACCCUUCAUU 264 A-138497.1 AAUGAAGGGUACGUUGUCA 515 683 683-701 AD-68854.1 A-138498.1 CCUUCAUUGAUGCCAAAAC 265 A-138499.1 GUUUUGGCAUCAAUGAAGG 516 694 694-712 AD-68855.1 A-138500.1 UGCCAAAACAACCAUCACA 266 A-138501.1 UGUGAUGGUUGUUUUGGCA 517 704 704-722 AD-68856.1 A-138502.1 AUCACCGUGUCCCCCUUCU 267 A-138503.1 AGAAGGGGGACACGGUGAU 518 717 717-735 AD-68857.1 A-138504.1 UCCCCCUUCUAUGGGGAGU 268 A-138505.1 ACUCCCCAUAGAAGGGGGA 519 726 726-744 AD-68858.1 A-138506.1 UGGGGAGUACGACAUCUGA 269 A-138507.1 UCAGAUGUCGUACUCCCCA 520 737 737-755 AD-68859.1 A-138508.1 AUCUGCCCUAAAGUCAAGU 270 A-138509.1 ACUUGACUUUAGGGCAGAU 521 750 750-768 AD-68860.1 A-138510.1 AGUCAAGUCCACGAACUUU 271 A-138511.1 AAAGUUCGUGGACUUGACU 522 761 761-779 AD-68861.1 A-138512.1 ACGAACUUUCUUCAUGUGA 272 A-138513.1 UCACAUGAAGAAAGUUCGU 523 771 771-789 AD-68862.1 A-138514.1 UUCAUGUGGACAUCACCAA 273 A-138515.1 UUGGUGAUGUCCACAUGAA 524 781 781-799 AD-68863.1 A-138516.1 UCACCAAGCUCAGUCUACA 274 A-138517.1 UGUAGACUGAGCUUGGUGA 525 793 793-811 AD-68864.1 A-138518.1 AGUCUACGCCUCUGCACAA 275 A-138519.1 UUGUGCAGAGGCGUAGACU 526 804 804-822 AD-68865.1 A-138520.1 CUGCACAGGGAACCUCUAA 276 A-138521.1 UUAGAGGUUCCCUGUGCAG 527 815 815-833 AD-68866.1 A-138522.1 AACCUCUACCUUCUCUCGA 277 A-138523.1 UCGAGAGAAGGUAGAGGUU 528 825 825-843 AD-68867.1 A-138524.1 UCUCGAGAGCUUUUGUCCA 278 A-138525.1 UGGACAAAAGCUCUCGAGA 529 838 838-856 AD-68868.1 A-138526.1 UUUGUCCCCCCGGAUCUCA 279 A-138527.1 UGAGAUCCGGGGGGACAAA 530 849 849-867 AD-68869.1 A-138528.1 CCGGAUCUCAAGGUGCUGA 280 A-138529.1 UCAGCACCUUGAGAUCCGG 531 858 858-876 AD-68870.1 A-138530.1 UGCUGGGAGAGAUAUGCCU 281 A-138531.1 AGGCAUAUCUCUCCCAGCA 532 871 871-889 AD-68871.1 A-138532.1 AGAUAUGCCUUCGAGGAUA 282 A-138533.1 UAUCCUCGAAGGCAUAUCU 533 880 880-898 AD-68872.1 A-138534.1 AGGAUAUUUGGAUGCAUUA 283 A-138535.1 UAAUGCAUCCAAAUAUCCU 534 893 893-911 AD-68873.1 A-138536.1 AUGCAUUCAGGUUCUUGGA 284 A-138537.1 UCCAAGAACCUGAAUGCAU 535 904 904-922 AD-68874.1 A-138538.1 UUCUUGGAAGAGAAGGGCA 285 A-138539.1 UGCCCUUCUCUUCCAAGAA 536 915 915-933 AD-68875.1 A-138540.1 GAGAAGGGCAUCUGCAACA 286 A-138541.1 UGUUGCAGAUGCCCUUCUC 537 924 924-942 AD-68876.1 A-138542.1 UGCAACAGGCCCCAGCCAA 287 A-138543.1 UUGGCUGGGGCCUGUUGCA 538 936 936-954 AD-68877.1 A-138544.1 CAGCCAGGCCUGAAGUCAU 288 A-138545.1 AUGACUUCAGGCCUGGCUG 539 948 948-966 AD-68878.1 A-138546.1 GAAGUCAUCCUCAGAAGGA 289 A-138547.1 UCCUUCUGAGGAUGACUUC 540 959 959-977 AD-68879.1 A-138548.1 UCAGAAGGGAUGGAUCCUA 290 A-138549.1 UAGGAUCCAUCCCUUCUGA 541 969 969-987 AD-68880.1 A-138550.1 UGGAUCCUGAGGUCGCCAU 291 A-138551.1 AUGGCGACCUCAGGAUCCA 542 979 979-997 AD-68881.1 A-138552.1 CGCCAUGCCCAGCUGGGCA 292 A-138553.1 UGCCCAGCUGGGCAUGGCG 543 992 992-1010 AD-68882.1 A-138554.1 CAGCUGGGCAAACAUGAGU 293 A-138555.1 ACUCAUGUUUGCCCAGCUG 544 1001 1001-1019 AD-68883.1 A-138556.1 CAUGAGUCUGGAUUCUUCA 294 A-138557.1 UGAAGAAUCCAGACUCAUG 545 1013 1013-1031 AD-68884.1 A-138558.1 UUCUUCCCCGGAGUCGGCU 295 A-138559.1 AGCCGACUCCGGGGAAGAA 546 1025 1025-1043 AD-68885.1 A-138560.1 AGUCGGCUGCCUUGGCUGU 296 A-138561.1 ACAGCCAAGGCAGCCGACU 547 1036 1036-1054 AD-68905.1 A-138562.1 UUGGCUGUGAGGCUGGAGA 297 A-138563.1 UCUCCAGCCUCACAGCCAA 548 1047 1047-1065 AD-68906.1 A-138564.1 AGGCUGGAGGGAGAUGAGA 298 A-138565.1 UCUCAUCUCCCUCCAGCCU 549 1056 1056-1074 AD-68907.1 A-138566.1 AUGAGCUGCUAGACCACCU 299 A-138567.1 AGGUGGUCUAGCAGCUCAU 550 1069 1069-1087 AD-68908.1 A-138568.1 UAGACCACCUGCGUCUCAA 300 A-138569.1 UUGAGACGCAGGUGGUCUA 551 1078 1078-1096 AD-68909.1 A-138570.1 CGUCUCAGCAUCCUGCCCU 301 A-138571.1 AGGGCAGGAUGCUGAGACG 552 1089 1089-1107 AD-68910.1 A-138572.1 CCUGCCCUGGGAUGAGAGA 302 A-138573.1 UCUCUCAUCCCAGGGCAGG 553 1100 1100-1118 AD-68911.1 A-138574.1 AUGAGAGCAUCCUGGACAA 303 A-138575.1 UUGUCCAGGAUGCUCUCAU 554 1111 1111-1129 AD-68912.1 A-138576.1 UGGACACCCUCUCGCCCAA 304 A-138577.1 UUGGGCGAGAGGGUGUCCA 555 1123 1123-1141 AD-68913.1 A-138578.1 UCGCCCAGGCUCGCUACAA 305 A-138579.1 UUGUAGCGAGCCUGGGCGA 556 1134 1134-1152 AD-68914.1 A-138580.1 UCGCUACAGCACUGAGUGA 306 A-138581.1 UCACUCAGUGCUGUAGCGA 557 1144 1144-1162 AD-68915.1 A-138582.1 CUGAGUGAAGAAAUGAAAG 307 A-138583.1 CUUUCAUUUCUUCACUCAG 558 1155 1155-1173 AD-68916.1 A-138584.1 AUGAAAGACAAAGGUGGAU 308 A-138585.1 AUCCACCUUUGUCUUUCAU 559 1167 1167-1185 AD-68917.1 A-138586.1 AAGGUGGAUACAUGAGCAA 309 A-138587.1 UUGCUCAUGUAUCCACCUU 560 1177 1177-1195 AD-68918.1 A-138588.1 AUGAGCAAGAUUUGCAACU 310 A-138589.1 AGUUGCAAAUCUUGCUCAU 561 1188 1188-1206 AD-68919.1 A-138590.1 UUGCAACUUGCUACCCAUU 311 A-138591.1 AAUGGGUAGCAAGUUGCAA 562 1199 1199-1217 AD-68920.1 A-138592.1 ACCCAUUAGGAUAAUGUCU 312 A-138593.1 AGACAUUAUCCUAAUGGGU 563 1211 1211-1229 AD-68921.1 A-138594.1 UAAUGUCUUAUGUAAUGCU 313 A-138595.1 AGCAUUACAUAAGACAUUA 564 1222 1222-1240 AD-68922.1 A-138596.1 UAAUGCUGCCCUGUACCCU 314 A-138597.1 AGGGUACAGGGCAGCAUUA 565 1234 1234-1252 AD-68923.1 A-138598.1 UGUACCCUGCCUGUGGAAU 315 A-138599.1 AUUCCACAGGCAGGGUACA 566 1245 1245-1263 AD-68924.1 A-138600.1 UGUGGAAUCUGCCAUUGCA 316 A-138601.1 UGCAAUGGCAGAUUCCACA 567 1256 1256-1274 AD-68925.1 A-138602.1 UGCCAUUGCGAUUGUCCAA 317 A-138603.1 UUGGACAAUCGCAAUGGCA 568 1265 1265-1283 AD-68926.1 A-138604.1 UUGUCCAGAGACUGGUGAA 318 A-138605.1 UUCACCAGUCUCUGGACAA 569 1276 1276-1294 AD-68927.1 A-138606.1 GGUGACAUGGCUUCCAGAU 319 A-138607.1 AUCUGGAAGCCAUGUCACC 570 1289 1289-1307 AD-68928.1 A-138608.1 UUCCAGAUAUGCCCGACGA 320 A-138609.1 UCGUCGGGCAUAUCUGGAA 571 1300 1300-1318 AD-68929.1 A-138610.1 UGCCCGACGAUGUCCUGUA 321 A-138611.1 UACAGGACAUCGUCGGGCA 572 1309 1309-1327 AD-68930.1 A-138612.1 UCCUGUGGUUGCAGUGGGU 322 A-138613.1 ACCCACUGCAACCACAGGA 573 1321 1321-1339 AD-68931.1 A-138614.1 AGUGGGUGACCUCACAGGU 323 A-138615.1 ACCUGUGAGGUCACCCACU 574 1333 1333-1351 AD-68932.1 A-138616.1 UCACAGGUGUUCACUCGAA 324 A-138617.1 UUCGAGUGAACACCUGUGA 575 1344 1344-1362 AD-68933.1 A-138618.1 UUCACUCGAGUGCUGAUGU 325 A-138619.1 ACAUCAGCACUCGAGUGAA 576 1353 1353-1371 AD-68934.1 A-138620.1 UGAUGUGUCUGCUCCCCGA 326 A-138621.1 UCGGGGAGCAGACACAUCA 577 1366 1366-1384 AD-68935.1 A-138622.1 UGCUCCCCGCCUCCAGGUA 327 A-138623.1 UACCUGGAGGCGGGGAGCA 578 1375 1375-1393 AD-68936.1 A-138624.1 CCAGGUCCCAAAUGCCAGU 328 A-138625.1 ACUGGCAUUUGGGACCUGG 579 1387 1387-1405 AD-68937.1 A-138626.1 AAUGCCAGUGAGCAGCCAA 329 A-138627.1 UUGGCUGCUCACUGGCAUU 580 1397 1397-1415 AD-68938.1 A-138628.1 AGCCAACAGGCCUCCCCAU 330 A-138629.1 AUGGGGAGGCCUGUUGGCU 581 1410 1410-1428 AD-68939.1 A-138630.1 CCUCCCCAUGCACACCUGA 331 A-138631.1 UCAGGUGUGCAUGGGGAGG 582 1420 1420-1438 AD-68940.1 A-138632.1 CACCUGAGCAGGACUGGCA 332 A-138633.1 UGCCAGUCCUGCUCAGGUG 583 1432 1432-1450 AD-68941.1 A-138634.1 GACUGGCCCUGCUGGACUA 333 A-138635.1 UAGUCCAGCAGGGCCAGUC 584 1443 1443-1461 AD-68942.1 A-138636.1 UGCUGGACUCCCUGCUCCA 334 A-138637.1 UGGAGCAGGGAGUCCAGCA 585 1452 1452-1470 AD-68943.1 A-138638.1 CUGCUCCCCCAAGGGCUGU 335 A-138639.1 ACAGCCCUUGGGGGAGCAG 586 1463 1463-1481 AD-68944.1 A-138640.1 AGGGCUGUCCAGCAGAGAA 336 A-138641.1 UUCUCUGCUGGACAGCCCU 587 1474 1474-1492 AD-68945.1 A-138642.1 CAGAGACCAAAGCAGAGGA 337 A-138643.1 UCCUCUGCUUUGGUCUCUG 588 1486 1486-1504 AD-68946.1 A-138644.1 AGCAGAGGCCACCCCGCGA 338 A-138645.1 UCGCGGGGUGGCCUCUGCU 589 1496 1496-1514 AD-68947.1 A-138646.1 CCGCGGUCCAUCCUCAGGU 339 A-138647.1 ACCUGAGGAUGGACCGCGG 590 1509 1509-1527 AD-68948.1 A-138648.1 CCUCAGGUCCAGCCUGAAA 340 A-138649.1 UUUCAGGCUGGACCUGAGG 591 1520 1520-1538 AD-68949.1 A-138650.1 AGCCUGAACUUCUUCUUGA 341 A-138651.1 UCAAGAAGAAGUUCAGGCU 592 1530 1530-1548 AD-68950.1 A-138652.1 UUCUUGGGCAAUAAAGUAA 342 A-138653.1 UUACUUUAUUGCCCAAGAA 593 1542 1542-1560 AD-68951.1 A-138654.1 UAAAGUACCUGCUGGUGCU 343 A-138655.1 AGCACCAGCAGGUACUUUA 594 1553 1553-1571 AD-68952.1 A-138656.1 CUGGUGCUGAGGGGCUCUA 344 A-138657.1 UAGAGCCCCUCAGCACCAG 595 1564 1564-1582 AD-68953.1 A-138658.1 AGGGGCUCUCCACCUUUCA 345 A-138659.1 UGAAAGGUGGAGAGCCCCU 596 1573 1573-1591 AD-68954.1 A-138660.1 CCUUUCCCAGUUUUUCACU 346 A-138661.1 AGUGAAAAACUGGGAAAGG 597 1585 1585-1603 AD-68955.1 A-138662.1 UUUUUCACUAGAGAAGAGU 347 A-138663.1 ACUCUUCUCUAGUGAAAAA 598 1595 1595-1613 AD-68956.1 A-138664.1 AAGAGUCUGUGAGUCACUU 348 A-138665.1 AAGUGACUCACAGACUCUU 599 1608 1608-1626 AD-68957.1 A-138666.1 AGUCACUUGAGGAGGCGAA 349 A-138667.1 UUCGCCUCCUCAAGUGACU 600 1619 1619-1637 AD-68958.1 A-138668.1 AGGAGGCGAGUCUAGCAGA 350 A-138669.1 UCUGCUAGACUCGCCUCCU 601 1628 1628-1646 AD-68959.1 A-138670.1 AGCAGAUUCUUUCAGAGGU 351 A-138671.1 ACCUCUGAAAGAAUCUGCU 602 1641 1641-1659 AD-68960.1 A-138672.1 UUCAGAGGUGCUAAAGUUU 352 A-138673.1 AAACUUUAGCACCUCUGAA 603 1651 1651-1669 AD-68961.1 A-138674.1 UAAAGUUUCCCAUCUUUGU 353 A-138675.1 ACAAAGAUGGGAAACUUUA 604 1662 1662-1680 AD-68962.1 A-138676.1 UCUUUGUGCAGCUACCUCA 354 A-138677.1 UGAGGUAGCUGCACAAAGA 605 1674 1674-1692 AD-68963.1 A-138678.1 AGCUACCUCCGCAUUGCUA 355 A-138679.1 UAGCAAUGCGGAGGUAGCU 606 1683 1683-1701 AD-68964.1 A-138680.1 UUGCUGUGUAGUGACCCCU 356 A-138681.1 AGGGGUCACUACACAGCAA 607 1696 1696-1714 AD-68965.1 A-138682.1 UGACCCCUGCCUGUGACGU 357 A-138683.1 ACGUCACAGGCAGGGGUCA 608 1707 1707-1725 AD-68966.1 A-138684.1 UGUGACGUGGAGGAUCCCA 358 A-138685.1 UGGGAUCCUCCACGUCACA 609 1718 1718-1736 AD-68967.1 A-138686.1 AGGAUCCCAGCCUCUGAGA 359 A-138687.1 UCUCAGAGGCUGGGAUCCU 610 1728 1728-1746 AD-68968.1 A-138688.1 CUCUGAGCUGAGUUGGUUU 360 A-138689.1 AAACCAACUCAGCUCAGAG 611 1739 1739-1757 AD-68969.1 A-138690.1 UUGGUUUUAUGAAAAGCUA 361 A-138691.1 UAGCUUUUCAUAAAACCAA 612 1751 1751-1769 AD-68970.1 A-138692.1 AAAAGCUAGGAAGCAACCU 362 A-138693.1 AGGUUGCUUCCUAGCUUUU 613 1762 1762-1780 AD-68971.1 A-138694.1 GAAGCAACCUUUCGCCUGU 363 A-138695.1 ACAGGCGAAAGGUUGCUUC 614 1771 1771-1789 AD-68972.1 A-138696.1 UCGCCUGUGCAGCGGUCCA 364 A-138697.1 UGGACCGCUGCACAGGCGA 615 1782 1782-1800 AD-68973.1 A-138698.1 CGGUCCAGCACUUAACUCU 365 A-138699.1 AGAGUUAAGUGCUGGACCG 616 1794 1794-1812 AD-68974.1 A-138700.1 UUAACUCUAAUACAUCAGA 366 A-138701.1 UCUGAUGUAUUAGAGUUAA 617 1805 1805-1823 AD-68975.1 A-138702.1 UACAUCAGCAUGCGUUAAU 367 A-138703.1 AUUAACGCAUGCUGAUGUA 618 1815 1815-1833 AD-68976.1 A-138704.1 CGUUAAUUCAGCUGGUUGA 368 A-138705.1 UCAACCAGCUGAAUUAACG 619 1827 1827-1845 AD-68977.1 A-138706.1 CUGGUUGGGAAAUGACACA 369 A-138707.1 UGUGUCAUUUCCCAACCAG 620 1838 1838-1856 AD-68978.1 A-138708.1 AAUGACACCAGGAAGCCCA 370 A-138709.1 UGGGCUUCCUGGUGUCAUU 621 1848 1848-1866 AD-68979.1 A-138710.1 AAGCCCAGUGCAGAGGGUA 371 A-138711.1 UACCCUCUGCACUGGGCUU 622 1860 1860-1878 AD-68980.1 A-138712.1 AGAGGGUCCCUUACUGACU 372 A-138713.1 AGUCAGUAAGGGACCCUCU 623 1871 1871-1889 AD-68981.1 A-138714.1 UUACUGACUGUUUCGUGGA 373 A-138715.1 UCCACGAAACAGUCAGUAA 624 1881 1881-1899 AD-68982.1 A-138716.1 UUCGUGGCCCUAUUAAUGA 374 A-138717.1 UCAUUAAUAGGGCCACGAA 625 1892 1892-1910 AD-68983.1 A-138718.1 UUAAUGGUCAGACUGUUCA 375 A-138719.1 UGAACAGUCUGACCAUUAA 626 1904 1904-1922 AD-68984.1 A-138720.1 GACUGUUCCAGCAUGAGGU 376 A-138721.1 ACCUCAUGCUGGAACAGUC 627 1914 1914-1932 AD-68985.1 A-138722.1 UGAGGUUCUUAGAAUGACA 377 A-138723.1 UGUCAUUCUAAGAACCUCA 628 1927 1927-1945 AD-68986.1 A-138724.1 UAGAAUGACAGGUGUUUGA 378 A-138725.1 UCAAACACCUGUCAUUCUA 629 1936 1936-1954 AD-68987.1 A-138726.1 UGUUUGGAUGGGUGGGGGA 379 A-138727.1 UCCCCCACCCAUCCAAACA 630 1948 1948-1966 AD-68988.1 A-138728.1 UGGGGGCCUUGUGAUGGGA 380 A-138729.1 UCCCAUCACAAGGCCCCCA 631 1960 1960-1978 AD-68989.1 A-138730.1 UGUGAUGGGGGGUAGGCUA 381 A-138731.1 UAGCCUACCCCCCAUCACA 632 1969 1969-1987 AD-68990.1 A-138732.1 UAGGCUGGCCCAUGUGUGA 382 A-138733.1 UCACACAUGGGCCAGCCUA 633 1981 1981-1999 AD-68991.1 A-138734.1 UGUGUGAUCUUGUGGGGUA 383 A-138735.1 UACCCCACAAGAUCACACA 634 1993 1993-2011 AD-68992.1 A-138736.1 UUGUGGGGUGGAGGGAAGA 384 A-138737.1 UCUUCCCUCCACCCCACAA 635 2002 2002-2020 AD-68993.1 A-138738.1 AGGGAAGAGAAUAGCAUGA 385 A-138739.1 UCAUGCUAUUCUCUUCCCU 636 2013 2013-2031 AD-68994.1 A-138740.1 UAGCAUGAUCCCACUUCCA 386 A-138741.1 UGGAAGUGGGAUCAUGCUA 637 2024 2024-2042 AD-68995.1 A-138742.1 ACUUCCCCAUGCUGUGGGA 387 A-138743.1 UCCCACAGCAUGGGGAAGU 638 2036 2036-2054 AD-68996.1 A-138744.1 CUGUGGGAAGGGGUGCAGU 388 A-138745.1 ACUGCACCCCUUCCCACAG 639 2047 2047-2065 AD-68997.1 A-138746.1 GUGCAGUUCGUCCCCAAGA 389 A-138747.1 UCUUGGGGACGAACUGCAC 640 2059 2059-2077 AD-68998.1 A-138748.1 UCCCCAAGAACGACACUGA 390 A-138749.1 UCAGUGUCGUUCUUGGGGA 641 2069 2069-2087 AD-69014.1 A-138750.1 ACACUGCCUGUCAGGUGGU 391 A-138751.1 ACCACCUGACAGGCAGUGU 642 2081 2081-2099 AD-69015.1 A-138752.1 UCAGGUGGUCUGCAAAGAU 392 A-138753.1 AUCUUUGCAGACCACCUGA 643 2091 2091-2109 AD-69016.1 A-138754.1 UGCAAAGAUGAUAACCUUA 393 A-138755.1 UAAGGUUAUCAUCUUUGCA 644 2101 2101-2119 AD-69017.1 A-138756.1 AACCUUGACUACUAAAAAC 394 A-138757.1 GUUUUUAGUAGUCAAGGUU 645 2113 2113-2131 AD-69018.1 A-138758.1 UAAAAACGUCUCCAUGGCA 395 A-138759.1 UGCCAUGGAGACGUUUUUA 646 2125 2125-2143 AD-69019.1 A-138760.1 CCAUGGCGGGGGUAACAAA 396 A-138761.1 UUUGUUACCCCCGCCAUGG 647 2136 2136-2154 AD-69020.1 A-138762.1 GGUAACAAGAUGAUAAUCU 397 A-138763.1 AGAUUAUCAUCUUGUUACC 648 2146 2146-2164 AD-69021.1 A-138764.1 UGAUAAUCUACUUAAUUUU 398 A-138765.1 AAAAUUAAGUAGAUUAUCA 649 2156 2156-2174 AD-69022.1 A-138766.1 UUAAUUUUAGAACACCUUU 399 A-138767.1 AAAGGUGUUCUAAAAUUAA 650 2167 2167-2185 AD-69023.1 A-138768.1 ACACCUUUUUCACCUAACU 400 A-138769.1 AGUUAGGUGAAAAAGGUGU 651 2178 2178-2196 AD-69024.1 A-138770.1 CCUAACUAAAAUAAUGUUU 401 A-138771.1 AAACAUUAUUUUAGUUAGG 652 2190 2190-2208 AD-69025.1 A-138772.1 AUAAUGUUUAAAGAGUUUU 402 A-138773.1 AAAACUCUUUAAACAUUAU 653 2200 2200-2218 AD-69026.1 A-138774.1 GAGUUUUGUAUAAAAAUGU 403 A-138775.1 ACAUUUUUAUACAAAACUC 654 2212 2212-2230 AD-69027.1 A-138776.1 AAAAAUGUAAGGAAGCGUU 404 A-138777.1 AACGCUUCCUUACAUUUUU 655 2223 2223-2241 AD-69028.1 A-138778.1 GGAAGCGUUGUUACCUGUU 405 A-138779.1 AACAGGUAACAACGCUUCC 656 2233 2233-2251 AD-69029.1 A-138780.1 UACCUGUUGAAUUUUGUAU 406 A-138781.1 AUACAAAAUUCAACAGGUA 657 2244 2244-2262 AD-69030.1 A-138782.1 UUGUAUUAUGUGAAUCAGU 407 A-138783.1 ACUGAUUCACAUAAUACAA 658 2257 2257-2275 AD-69031.1 A-138784.1 GAAUCAGUGAGAUGUUAGU 408 A-138785.1 ACUAACAUCUCACUGAUUC 659 2268 2268-2286 AD-69032.1 A-138786.1 AUGUUAGUAGAAUAAGCCU 409 A-138787.1 AGGCUUAUUCUACUAACAU 660 2279 2279-2297 AD-69033.1 A-138788.1 AUAAGCCUUAAAAAAAAAA 410 A-138789.1 UUUUUUUUUUAAGGCUUAU 661 2290 2290-2308 AD-69034.1 A-138790.1 AAAAAAAAAAAAAUCGGUU 411 A-138791.1 AACCGAUUUUUUUUUUUUU 662 2299 2299-2317 AD-69035.1 A-138792.1 AAUCGGUUGGGUGCAGUGA 412 A-138793.1 UCACUGCACCCAACCGAUU 663 2310 2310-2328 AD-69036.1 A-138794.1 UGCAGUGGCACACGGCUGU 413 A-138795.1 ACAGCCGUGUGCCACUGCA 664 2321 2321-2339 AD-69037.1 A-138796.1 GGCUGUAAUCCCAGCACUU 414 A-138797.1 AAGUGCUGGGAUUACAGCC 665 2334 2334-2352 AD-69038.1 A-138798.1 CAGCACUUUGGGAGGCCAA 415 A-138799.1 UUGGCCUCCCAAAGUGCUG 666 2345 2345-2363 AD-69039.1 A-138800.1 GAGGCCAAGGUUGGCAGAU 416 A-138801.1 AUCUGCCAACCUUGGCCUC 667 2356 2356-2374 AD-69040.1 A-138802.1 UUGGCAGAUCACCUGAGGU 417 A-138803.1 ACCUCAGGUGAUCUGCCAA 668 2366 2366-2384 AD-69041.1 A-138804.1 CUGAGGUCAGGAGUUCAAA 418 A-138805.1 UUUGAACUCCUGACCUCAG 669 2378 2378-2396 AD-69042.1 A-138806.1 GAGUUCAAGACCAGUCUGA 419 A-138807.1 UCAGACUGGUCUUGAACUC 670 2388 2388-2406 AD-69043.1 A-138808.1 CAGUCUGGCCAACAUAGCA 420 A-138809.1 UGCUAUGUUGGCCAGACUG 671 2399 2399-2417 AD-69044.1 A-138810.1 AACAUAGCAAAACCCUGUA 421 A-138811.1 UACAGGGUUUUGCUAUGUU 672 2409 2409-2427 AD-69045.1 A-138812.1 CCCUGUCUCUACUAAAAAU 422 A-138813.1 AUUUUUAGUAGAGACAGGG 673 2421 2421-2439 AD-69046.1 A-138814.1 CUAAAAAUACAAAAAUUAU 423 A-138815.1 AUAAUUUUUGUAUUUUUAG 674 2432 2432-2450 AD-69047.1 A-138816.1 AAAAUUAUCUGGGCAUGGU 424 A-138817.1 ACCAUGCCCAGAUAAUUUU 675 2443 2443-2461 AD-69048.1 A-138818.1 GGCAUGGUGGUGCAUGCCU 425 A-138819.1 AGGCAUGCACCACCAUGCC 676 2454 2454-2472 AD-69049.1 A-138820.1 CAUGCCUGUAAUCCCAGCU 426 A-138821.1 AGCUGGGAUUACAGGCAUG 677 2466 2466-2484 AD-69050.1 A-138822.1 AAUCCCAGCUAUUCGGAAA 427 A-138823.1 UUUCCGAAUAGCUGGGAUU 678 2475 2475-2493 AD-69051.1 A-138824.1 UUCGGAAGGCUGAGGCAGA 428 A-138825.1 UCUGCCUCAGCCUUCCGAA 679 2486 2486-2504 AD-69052.1 A-138826.1 AGGCAGGAGAAUCACUUGA 429 A-138827.1 UCAAGUGAUUCUCCUGCCU 680 2498 2498-2516 AD-69053.1 A-138828.1 AUCACUUGAACCCAGGAGA 430 A-138829.1 UCUCCUGGGUUCAAGUGAU 681 2508 2508-2526 AD-69054.1 A-138830.1 CAGGAGGCGGAGGUUGCGA 431 A-138831.1 UCGCAACCUCCGCCUCCUG 682 2520 2520-2538 AD-69055.1 A-138832.1 GUUGCGGUGAGCUGAGAUU 432 A-138833.1 AAUCUCAGCUCACCGCAAC 683 2532 2532-2550 AD-69056.1 A-138834.1 CUGAGAUUGCACCAUUUCA 433 A-138835.1 UGAAAUGGUGCAAUCUCAG 684 2543 2543-2561 AD-69057.1 A-138836.1 CACCAUUUCAUUCCAGCCU 434 A-138837.1 AGGCUGGAAUGAAAUGGUG 685 2552 2552-2570 AD-69058.1 A-138838.1 CAGCCUGGGCAACAUGAGU 435 A-138839.1 ACUCAUGUUGCCCAGGCUG 686 2565 2565-2583 AD-69059.1 A-138840.1 AACAUGAGUGAAAGUCUGA 436 A-138841.1 UCAGACUUUCACUCAUGUU 687 2575 2575-2593 AD-69060.1 A-138842.1 AGUCUGACUCAAAAAAAAA 437 A-138843.1 UUUUUUUUUGAGUCAGACU 688 2587 2587-2605 AD-69061.1 A-138844.1 AAAAAAAAAAAAUUUAAAA 438 A-138845.1 UUUUAAAUUUUUUUUUUUU 689 2597 2597-2615 AD-69062.1 A-138846.1 UUUAAAAAACAAAAUAAUA 439 A-138847.1 UAUUAUUUUGUUUUUUAAA 690 2609 2609-2627 AD-69063.1 A-138848.1 AAAAUAAUCUAGUGUGCAA 440 A-138849.1 UUGCACACUAGAUUAUUUU 691 2619 2619-2637 AD-69064.1 A-138850.1 GUGUGCAGGGCAUUCACCU 441 A-138851.1 AGGUGAAUGCCCUGCACAC 692 2630 2630-2648 AD-69065.1 A-138852.1 CAUUCACCUCAGCCCCCCA 442 A-138853.1 UGGGGGGCUGAGGUGAAUG 693 2640 2640-2658 AD-69066.1 A-138854.1 CCCCCAGGCAGGAGCCAAA 443 A-138855.1 UUUGGCUCCUGCCUGGGGG 694 2653 2653-2671 AD-69067.1 A-138856.1 AGGAGCCAAGCACAGCAGA 444 A-138857.1 UCUGCUGUGCUUGGCUCCU 695 2662 2662-2680 AD-69068.1 A-138858.1 ACAGCAGGAGCUUCCGCCU 445 A-138859.1 AGGCGGAAGCUCCUGCUGU 696 2673 2673-2691 AD-69069.1 A-138860.1 UUCCGCCUCCUCUCCACUA 446 A-138861.1 UAGUGGAGAGGAGGCGGAA 697 2684 2684-2702 AD-69070.1 A-138862.1 UCCACUGGAGCACACAACU 447 A-138863.1 AGUUGUGUGCUCCAGUGGA 698 2696 2696-2714 AD-69071.1 A-138864.1 ACACAACUUGAACCUGGCU 448 A-138865.1 AGCCAGGUUCAAGUUGUGU 699 2707 2707-2725 AD-69072.1 A-138866.1 AACCUGGCUUAUUUUCUGA 449 A-138867.1 UCAGAAAAUAAGCCAGGUU 700 2717 2717-2735 AD-69073.1 A-138868.1 UUCUGCAGGGACCAGCCCA 450 A-138869.1 UGGGCUGGUCCCUGCAGAA 701 2730 2730-2748 AD-69074.1 A-138870.1 CCAGCCCCACAUGGUCAGU 451 A-138871.1 ACUGACCAUGUGGGGCUGG 702 2741 2741-2759 AD-69076.1 A-138874.1 UUUCUCCCCAUGUGUGGCA 452 A-138875.1 UGCCACACAUGGGGAGAAA 703 2763 2763-2781 AD-69077.1 A-138878.1 AGAGAGUGUAGAAAUAAAG 453 A-138879.1 CUUUAUUUCUACACUCUCU 704 2785 2785-2803

TABLE 5 Modified Sense and Antisense Strand Sequences of PNPLA3 RNAi Agents SEQ SEQ Sense Oligo ID Antisense ID Duplex Name Name Sense Sequence (5′-3′) NO Oligo Name Sequence (5′-3′) NO AD-68792.1 A-138374.1 GGGGGCGGGGCUGACGUCAdTdT 705 A-138375.1 UGACGUCAGCCCCGCCCCCdTdT 956 AD-68793.1 A-138376.1 ACGUCGCGCUGGGAAUGCAdTdT 706 A-138377.1 UGCAUUCCCAGCGCGACGUdTdT 957 AD-68794.1 A-138378.1 GGAAUGCCCUGGCCGAGAAdTdT 707 A-138379.1 UUCUCGGCCAGGGCAUUCCdTdT 958 AD-68795.1 A-138380.1 UGGCCGAGACACUGAGGCAdTdT 708 A-138381.1 UGCCUCAGUGUCUCGGCCAdTdT 959 AD-68796.1 A-138382.1 UGAGGCAGGGUAGAGAGCAdTdT 709 A-138383.1 UGCUCUCUACCCUGCCUCAdTdT 960 AD-68797.1 A-138384.1 AGAGAGCGCUUGCGGGCGAdTdT 710 A-138385.1 UCGCCCGCAAGCGCUCUCUdTdT 961 AD-68798.1 A-138386.1 CGGGCGCCGGGCGGAGCUAdTdT 711 A-138387.1 UAGCUCCGCCCGGCGCCCGdTdT 962 AD-68799.1 A-138388.1 GCGGAGCUGCUGCGGAUCAdTdT 712 A-138389.1 UGAUCCGCAGCAGCUCCGCdTdT 963 AD-68800.1 A-138390.1 UGCGGAUCAGGACCCGAGAdTdT 713 A-138391.1 UCUCGGGUCCUGAUCCGCAdTdT 964 AD-68801.1 A-138392.1 ACCCGAGCCGAUUCCCGAUdTdT 714 A-138393.1 AUCGGGAAUCGGCUCGGGUdTdT 965 AD-68802.1 A-138394.1 UUCCCGAUCCCGACCCAGAdTdT 715 A-138395.1 UCUGGGUCGGGAUCGGGAAdTdT 966 AD-68803.1 A-138396.1 ACCCAGAUCCUAACCCGCAdTdT 716 A-138397.1 UGCGGGUUAGGAUCUGGGUdTdT 967 AD-68804.1 A-138398.1 UAACCCGCGCCCCCGCCCAdTdT 717 A-138399.1 UGGGCGGGGGCGCGGGUUAdTdT 968 AD-68805.1 A-138400.1 CCGCCCCGCCGCCGCCGCAdTdT 718 A-138401.1 UGCGGCGGCGGCGGGGCGGdTdT 969 AD-68806.1 A-138402.1 CGCCGCCAUGUACGACGCAdTdT 719 A-138403.1 UGCGUCGUACAUGGCGGCGdTdT 970 AD-68807.1 A-138404.1 UACGACGCAGAGCGCGGCUdTdT 720 A-138405.1 AGCCGCGCUCUGCGUCGUAdTdT 971 AD-68808.1 A-138406.1 CGCGGCUGGAGCUUGUCCUdTdT 721 A-138407.1 AGGACAAGCUCCAGCCGCGdTdT 972 AD-68809.1 A-138408.1 AGCUUGUCCUUCGCGGGCUdTdT 722 A-138409.1 AGCCCGCGAAGGACAAGCUdTdT 973 AD-68810.1 A-138410.1 CGCGGGCUGCGGCUUCCUAdTdT 723 A-138411.1 UAGGAAGCCGCAGCCCGCGdTdT 974 AD-68811.1 A-138412.1 UUCCUGGGCUUCUACCACAdTdT 724 A-138413.1 UGUGGUAGAAGCCCAGGAAdTdT 975 AD-68812.1 A-138414.1 UUCUACCACGUCGGGGCGAdTdT 725 A-138415.1 UCGCCCCGACGUGGUAGAAdTdT 976 AD-68813.1 A-138416.1 CGGGGCGACCCGCUGCCUAdTdT 726 A-138417.1 UAGGCAGCGGGUCGCCCCGdTdT 977 AD-68814.1 A-138418.1 UGCCUGAGCGAGCACGCCAdTdT 727 A-138419.1 UGGCGUGCUCGCUCAGGCAdTdT 978 AD-68815.1 A-138420.1 AGCACGCCCCGCACCUCCUdTdT 728 A-138421.1 AGGAGGUGCGGGGCGUGCUdTdT 979 AD-68816.1 A-138422.1 ACCUCCUCCGCGACGCGCAdTdT 729 A-138423.1 UGCGCGUCGCGGAGGAGGUdTdT 980 AD-68817.1 A-138424.1 GCGACGCGCGCAUGUUGUUdTdT 730 A-138425.1 AACAACAUGCGCGCGUCGCdTdT 981 AD-68818.1 A-138426.1 UGUUGUUCGGCGCUUCGGAdTdT 731 A-138427.1 UCCGAAGCGCCGAACAACAdTdT 982 AD-68819.1 A-138428.1 CUUCGGCCGGGGCGUUGCAdTdT 732 A-138429.1 UGCAACGCCCCGGCCGAAGdTdT 983 AD-68820.1 A-138430.1 GGCGUUGCACUGCGUCGGAdTdT 733 A-138431.1 UCCGACGCAGUGCAACGCCdTdT 984 AD-68821.1 A-138432.1 UGCGUCGGCGUCCUCUCCAdTdT 734 A-138433.1 UGGAGAGGACGCCGACGCAdTdT 985 AD-68822.1 A-138434.1 UCUCCGGUAUCCCGCUGGAdTdT 735 A-138435.1 UCCAGCGGGAUACCGGAGAdTdT 986 AD-68823.1 A-138436.1 UCCCGCUGGAGCAGACUCUdTdT 736 A-138437.1 AGAGUCUGCUCCAGCGGGAdTdT 987 AD-68824.1 A-138438.1 CAGACUCUGCAGGUCCUCUdTdT 737 A-138439.1 AGAGGACCUGCAGAGUCUGdTdT 988 AD-68825.1 A-138440.1 UCCUCUCAGAUCUUGUGCAdTdT 738 A-138441.1 UGCACAAGAUCUGAGAGGAdTdT 989 AD-68826.1 A-138442.1 UCUUGUGCGGAAGGCCAGAdTdT 739 A-138443.1 UCUGGCCUUCCGCACAAGAdTdT 990 AD-68827.1 A-138444.1 AAGGCCAGGAGUCGGAACAdTdT 740 A-138445.1 UGUUCCGACUCCUGGCCUUdTdT 991 AD-68828.1 A-138446.1 CGGAACAUUGGCAUCUUCAdTdT 741 A-138447.1 UGAAGAUGCCAAUGUUCCGdTdT 992 AD-68829.1 A-138448.1 GCAUCUUCCAUCCAUCCUUdTdT 742 A-138449.1 AAGGAUGGAUGGAAGAUGCdTdT 993 AD-68830.1 A-138450.1 CCAUCCUUCAACUUAAGCAdTdT 743 A-138451.1 UGCUUAAGUUGAAGGAUGGdTdT 994 AD-68831.1 A-138452.1 UUAAGCAAGUUCCUCCGAAdTdT 744 A-138453.1 UUCGGAGGAACUUGCUUAAdTdT 995 AD-68832.1 A-138454.1 CCUCCGACAGGGUCUCUGAdTdT 745 A-138455.1 UCAGAGACCCUGUCGGAGGdTdT 996 AD-68833.1 A-138456.1 UCUCUGCAAAUGCCUCCCAdTdT 746 A-138457.1 UGGGAGGCAUUUGCAGAGAdTdT 997 AD-68834.1 A-138458.1 UGCCUCCCGGCCAAUGUCAdTdT 747 A-138459.1 UGACAUUGGCCGGGAGGCAdTdT 998 AD-68835.1 A-138460.1 AAUGUCCACCAGCUCAUCUdTdT 748 A-138461.1 AGAUGAGCUGGUGGACAUUdTdT 999 AD-68836.1 A-138462.1 AGCUCAUCUCCGGCAAAAUdTdT 749 A-138463.1 AUUUUGCCGGAGAUGAGCUdTdT 1000 AD-68837.1 A-138464.1 CGGCAAAAUAGGCAUCUCUdTdT 750 A-138465.1 AGAGAUGCCUAUUUUGCCGdTdT 1001 AD-68838.1 A-138466.1 AUCUCUCUUACCAGAGUGUdTdT 751 A-138467.1 ACACUCUGGUAAGAGAGAUdTdT 1002 AD-68839.1 A-138468.1 ACCAGAGUGUCUGAUGGGAdTdT 752 A-138469.1 UCCCAUCAGACACUCUGGUdTdT 1003 AD-68840.1 A-138470.1 AUGGGGAAAACGUUCUGGUdTdT 753 A-138471.1 ACCAGAACGUUUUCCCCAUdTdT 1004 AD-68841.1 A-138472.1 ACGUUCUGGUGUCUGACUUdTdT 754 A-138473.1 AAGUCAGACACCAGAACGUdTdT 1005 AD-68842.1 A-138474.1 UCUGACUUUCGGUCCAAAGdTdT 755 A-138475.1 CUUUGGACCGAAAGUCAGAdTdT 1006 AD-68843.1 A-138476.1 UCCAAAGACGAAGUCGUGAdTdT 756 A-138477.1 UCACGACUUCGUCUUUGGAdTdT 1007 AD-68844.1 A-138478.1 AAGUCGUGGAUGCCUUGGUdTdT 757 A-138479.1 ACCAAGGCAUCCACGACUUdTdT 1008 AD-68845.1 A-138480.1 CCUUGGUAUGUUCCUGCUUdTdT 758 A-138481.1 AAGCAGGAACAUACCAAGGdTdT 1009 AD-68846.1 A-138482.1 UCCUGCUUCAUCCCCUUCUdTdT 759 A-138483.1 AGAAGGGGAUGAAGCAGGAdTdT 1010 AD-68847.1 A-138484.1 UCCCCUUCUACAGUGGCCUdTdT 760 A-138485.1 AGGCCACUGUAGAAGGGGAdTdT 1011 AD-68848.1 A-138486.1 AGUGGCCUUAUCCCUCCUUdTdT 761 A-138487.1 AAGGAGGGAUAAGGCCACUdTdT 1012 AD-68849.1 A-138488.1 CCUCCUUCCUUCAGAGGCAdTdT 762 A-138489.1 UGCCUCUGAAGGAAGGAGGdTdT 1013 AD-68850.1 A-138490.1 UCAGAGGCGUGCGAUAUGUdTdT 763 A-138491.1 ACAUAUCGCACGCCUCUGAdTdT 1014 AD-68851.1 A-138492.1 GAUAUGUGGAUGGAGGAGUdTdT 764 A-138493.1 ACUCCUCCAUCCACAUAUCdTdT 1015 AD-68852.1 A-138494.1 GAGGAGUGAGUGACAACGUdTdT 765 A-138495.1 ACGUUGUCACUCACUCCUCdTdT 1016 AD-68853.1 A-138496.1 UGACAACGUACCCUUCAUUdTdT 766 A-138497.1 AAUGAAGGGUACGUUGUCAdTdT 1017 AD-68854.1 A-138498.1 CCUUCAUUGAUGCCAAAACdTdT 767 A-138499.1 GUUUUGGCAUCAAUGAAGGdTdT 1018 AD-68855.1 A-138500.1 UGCCAAAACAACCAUCACAdTdT 768 A-138501.1 UGUGAUGGUUGUUUUGGCAdTdT 1019 AD-68856.1 A-138502.1 AUCACCGUGUCCCCCUUCUdTdT 769 A-138503.1 AGAAGGGGGACACGGUGAUdTdT 1020 AD-68857.1 A-138504.1 UCCCCCUUCUAUGGGGAGUdTdT 770 A-138505.1 ACUCCCCAUAGAAGGGGGAdTdT 1021 AD-68858.1 A-138506.1 UGGGGAGUACGACAUCUGAdTdT 771 A-138507.1 UCAGAUGUCGUACUCCCCAdTdT 1022 AD-68859.1 A-138508.1 AUCUGCCCUAAAGUCAAGUdTdT 772 A-138509.1 ACUUGACUUUAGGGCAGAUdTdT 1023 AD-68860.1 A-138510.1 AGUCAAGUCCACGAACUUUdTdT 773 A-138511.1 AAAGUUCGUGGACUUGACUdTdT 1024 AD-68861.1 A-138512.1 ACGAACUUUCUUCAUGUGAdTdT 774 A-138513.1 UCACAUGAAGAAAGUUCGUdTdT 1025 AD-68862.1 A-138514.1 UUCAUGUGGACAUCACCAAdTdT 775 A-138515.1 UUGGUGAUGUCCACAUGAAdTdT 1026 AD-68863.1 A-138516.1 UCACCAAGCUCAGUCUACAdTdT 776 A-138517.1 UGUAGACUGAGCUUGGUGAdTdT 1027 AD-68864.1 A-138518.1 AGUCUACGCCUCUGCACAAdTdT 777 A-138519.1 UUGUGCAGAGGCGUAGACUdTdT 1028 AD-68865.1 A-138520.1 CUGCACAGGGAACCUCUAAdTdT 778 A-138521.1 UUAGAGGUUCCCUGUGCAGdTdT 1029 AD-68866.1 A-138522.1 AACCUCUACCUUCUCUCGAdTdT 779 A-138523.1 UCGAGAGAAGGUAGAGGUUdTdT 1030 AD-68867.1 A-138524.1 UCUCGAGAGCUUUUGUCCAdTdT 780 A-138525.1 UGGACAAAAGCUCUCGAGAdTdT 1031 AD-68868.1 A-138526.1 UUUGUCCCCCCGGAUCUCAdTdT 781 A-138527.1 UGAGAUCCGGGGGGACAAAdTdT 1032 AD-68869.1 A-138528.1 CCGGAUCUCAAGGUGCUGAdTdT 782 A-138529.1 UCAGCACCUUGAGAUCCGGdTdT 1033 AD-68870.1 A-138530.1 UGCUGGGAGAGAUAUGCCUdTdT 783 A-138531.1 AGGCAUAUCUCUCCCAGCAdTdT 1034 AD-68871.1 A-138532.1 AGAUAUGCCUUCGAGGAUAdTdT 784 A-138533.1 UAUCCUCGAAGGCAUAUCUdTdT 1035 AD-68872.1 A-138534.1 AGGAUAUUUGGAUGCAUUAdTdT 785 A-138535.1 UAAUGCAUCCAAAUAUCCUdTdT 1036 AD-68873.1 A-138536.1 AUGCAUUCAGGUUCUUGGAdTdT 786 A-138537.1 UCCAAGAACCUGAAUGCAUdTdT 1037 AD-68874.1 A-138538.1 UUCUUGGAAGAGAAGGGCAdTdT 787 A-138539.1 UGCCCUUCUCUUCCAAGAAdTdT 1038 AD-68875.1 A-138540.1 GAGAAGGGCAUCUGCAACAdTdT 788 A-138541.1 UGUUGCAGAUGCCCUUCUCdTdT 1039 AD-68876.1 A-138542.1 UGCAACAGGCCCCAGCCAAdTdT 789 A-138543.1 UUGGCUGGGGCCUGUUGCAdTdT 1040 AD-68877.1 A-138544.1 CAGCCAGGCCUGAAGUCAUdTdT 790 A-138545.1 AUGACUUCAGGCCUGGCUGdTdT 1041 AD-68878.1 A-138546.1 GAAGUCAUCCUCAGAAGGAdTdT 791 A-138547.1 UCCUUCUGAGGAUGACUUCdTdT 1042 AD-68879.1 A-138548.1 UCAGAAGGGAUGGAUCCUAdTdT 792 A-138549.1 UAGGAUCCAUCCCUUCUGAdTdT 1043 AD-68880.1 A-138550.1 UGGAUCCUGAGGUCGCCAUdTdT 793 A-138551.1 AUGGCGACCUCAGGAUCCAdTdT 1044 AD-68881.1 A-138552.1 CGCCAUGCCCAGCUGGGCAdTdT 794 A-138553.1 UGCCCAGCUGGGCAUGGCGdTdT 1045 AD-68882.1 A-138554.1 CAGCUGGGCAAACAUGAGUdTdT 795 A-138555.1 ACUCAUGUUUGCCCAGCUGdTdT 1046 AD-68883.1 A-138556.1 CAUGAGUCUGGAUUCUUCAdTdT 796 A-138557.1 UGAAGAAUCCAGACUCAUGdTdT 1047 AD-68884.1 A-138558.1 UUCUUCCCCGGAGUCGGCUdTdT 797 A-138559.1 AGCCGACUCCGGGGAAGAAdTdT 1048 AD-68885.1 A-138560.1 AGUCGGCUGCCUUGGCUGUdTdT 798 A-138561.1 ACAGCCAAGGCAGCCGACUdTdT 1049 AD-68905.1 A-138562.1 UUGGCUGUGAGGCUGGAGAdTdT 799 A-138563.1 UCUCCAGCCUCACAGCCAAdTdT 1050 AD-68906.1 A-138564.1 AGGCUGGAGGGAGAUGAGAdTdT 800 A-138565.1 UCUCAUCUCCCUCCAGCCUdTdT 1051 AD-68907.1 A-138566.1 AUGAGCUGCUAGACCACCUdTdT 801 A-138567.1 AGGUGGUCUAGCAGCUCAUdTdT 1052 AD-68908.1 A-138568.1 UAGACCACCUGCGUCUCAAdTdT 802 A-138569.1 UUGAGACGCAGGUGGUCUAdTdT 1053 AD-68909.1 A-138570.1 CGUCUCAGCAUCCUGCCCUdTdT 803 A-138571.1 AGGGCAGGAUGCUGAGACGdTdT 1054 AD-68910.1 A-138572.1 CCUGCCCUGGGAUGAGAGAdTdT 804 A-138573.1 UCUCUCAUCCCAGGGCAGGdTdT 1055 AD-68911.1 A-138574.1 AUGAGAGCAUCCUGGACAAdTdT 805 A-138575.1 UUGUCCAGGAUGCUCUCAUdTdT 1056 AD-68912.1 A-138576.1 UGGACACCCUCUCGCCCAAdTdT 806 A-138577.1 UUGGGCGAGAGGGUGUCCAdTdT 1057 AD-68913.1 A-138578.1 UCGCCCAGGCUCGCUACAAdTdT 807 A-138579.1 UUGUAGCGAGCCUGGGCGAdTdT 1058 AD-68914.1 A-138580.1 UCGCUACAGCACUGAGUGAdTdT 808 A-138581.1 UCACUCAGUGCUGUAGCGAdTdT 1059 AD-68915.1 A-138582.1 CUGAGUGAAGAAAUGAAAGdTdT 809 A-138583.1 CUUUCAUUUCUUCACUCAGdTdT 1060 AD-68916.1 A-138584.1 AUGAAAGACAAAGGUGGAUdTdT 810 A-138585.1 AUCCACCUUUGUCUUUCAUdTdT 1061 AD-68917.1 A-138586.1 AAGGUGGAUACAUGAGCAAdTdT 811 A-138587.1 UUGCUCAUGUAUCCACCUUdTdT 1062 AD-68918.1 A-138588.1 AUGAGCAAGAUUUGCAACUdTdT 812 A-138589.1 AGUUGCAAAUCUUGCUCAUdTdT 1063 AD-68919.1 A-138590.1 UUGCAACUUGCUACCCAUUdTdT 813 A-138591.1 AAUGGGUAGCAAGUUGCAAdTdT 1064 AD-68920.1 A-138592.1 ACCCAUUAGGAUAAUGUCUdTdT 814 A-138593.1 AGACAUUAUCCUAAUGGGUdTdT 1065 AD-68921.1 A-138594.1 UAAUGUCUUAUGUAAUGCUdTdT 815 A-138595.1 AGCAUUACAUAAGACAUUAdTdT 1066 AD-68922.1 A-138596.1 UAAUGCUGCCCUGUACCCUdTdT 816 A-138597.1 AGGGUACAGGGCAGCAUUAdTdT 1067 AD-68923.1 A-138598.1 UGUACCCUGCCUGUGGAAUdTdT 817 A-138599.1 AUUCCACAGGCAGGGUACAdTdT 1068 AD-68924.1 A-138600.1 UGUGGAAUCUGCCAUUGCAdTdT 818 A-138601.1 UGCAAUGGCAGAUUCCACAdTdT 1069 AD-68925.1 A-138602.1 UGCCAUUGCGAUUGUCCAAdTdT 819 A-138603.1 UUGGACAAUCGCAAUGGCAdTdT 1070 AD-68926.1 A-138604.1 UUGUCCAGAGACUGGUGAAdTdT 820 A-138605.1 UUCACCAGUCUCUGGACAAdTdT 1071 AD-68927.1 A-138606.1 GGUGACAUGGCUUCCAGAUdTdT 821 A-138607.1 AUCUGGAAGCCAUGUCACCdTdT 1072 AD-68928.1 A-138608.1 UUCCAGAUAUGCCCGACGAdTdT 822 A-138609.1 UCGUCGGGCAUAUCUGGAAdTdT 1073 AD-68929.1 A-138610.1 UGCCCGACGAUGUCCUGUAdTdT 823 A-138611.1 UACAGGACAUCGUCGGGCAdTdT 1074 AD-68930.1 A-138612.1 UCCUGUGGUUGCAGUGGGUdTdT 824 A-138613.1 ACCCACUGCAACCACAGGAdTdT 1075 AD-68931.1 A-138614.1 AGUGGGUGACCUCACAGGUdTdT 825 A-138615.1 ACCUGUGAGGUCACCCACUdTdT 1076 AD-68932.1 A-138616.1 UCACAGGUGUUCACUCGAAdTdT 826 A-138617.1 UUCGAGUGAACACCUGUGAdTdT 1077 AD-68933.1 A-138618.1 UUCACUCGAGUGCUGAUGUdTdT 827 A-138619.1 ACAUCAGCACUCGAGUGAAdTdT 1078 AD-68934.1 A-138620.1 UGAUGUGUCUGCUCCCCGAdTdT 828 A-138621.1 UCGGGGAGCAGACACAUCAdTdT 1079 AD-68935.1 A-138622.1 UGCUCCCCGCCUCCAGGUAdTdT 829 A-138623.1 UACCUGGAGGCGGGGAGCAdTdT 1080 AD-68936.1 A-138624.1 CCAGGUCCCAAAUGCCAGUdTdT 830 A-138625.1 ACUGGCAUUUGGGACCUGGdTdT 1081 AD-68937.1 A-138626.1 AAUGCCAGUGAGCAGCCAAdTdT 831 A-138627.1 UUGGCUGCUCACUGGCAUUdTdT 1082 AD-68938.1 A-138628.1 AGCCAACAGGCCUCCCCAUdTdT 832 A-138629.1 AUGGGGAGGCCUGUUGGCUdTdT 1083 AD-68939.1 A-138630.1 CCUCCCCAUGCACACCUGAdTdT 833 A-138631.1 UCAGGUGUGCAUGGGGAGGdTdT 1084 AD-68940.1 A-138632.1 CACCUGAGCAGGACUGGCAdTdT 834 A-138633.1 UGCCAGUCCUGCUCAGGUGdTdT 1085 AD-68941.1 A-138634.1 GACUGGCCCUGCUGGACUAdTdT 835 A-138635.1 UAGUCCAGCAGGGCCAGUCdTdT 1086 AD-68942.1 A-138636.1 UGCUGGACUCCCUGCUCCAdTdT 836 A-138637.1 UGGAGCAGGGAGUCCAGCAdTdT 1087 AD-68943.1 A-138638.1 CUGCUCCCCCAAGGGCUGUdTdT 837 A-138639.1 ACAGCCCUUGGGGGAGCAGdTdT 1088 AD-68944.1 A-138640.1 AGGGCUGUCCAGCAGAGAAdTdT 838 A-138641.1 UUCUCUGCUGGACAGCCCUdTdT 1089 AD-68945.1 A-138642.1 CAGAGACCAAAGCAGAGGAdTdT 839 A-138643.1 UCCUCUGCUUUGGUCUCUGdTdT 1090 AD-68946.1 A-138644.1 AGCAGAGGCCACCCCGCGAdTdT 840 A-138645.1 UCGCGGGGUGGCCUCUGCUdTdT 1091 AD-68947.1 A-138646.1 CCGCGGUCCAUCCUCAGGUdTdT 841 A-138647.1 ACCUGAGGAUGGACCGCGGdTdT 1092 AD-68948.1 A-138648.1 CCUCAGGUCCAGCCUGAAAdTdT 842 A-138649.1 UUUCAGGCUGGACCUGAGGdTdT 1093 AD-68949.1 A-138650.1 AGCCUGAACUUCUUCUUGAdTdT 843 A-138651.1 UCAAGAAGAAGUUCAGGCUdTdT 1094 AD-68950.1 A-138652.1 UUCUUGGGCAAUAAAGUAAdTdT 844 A-138653.1 UUACUUUAUUGCCCAAGAAdTdT 1095 AD-68951.1 A-138654.1 UAAAGUACCUGCUGGUGCUdTdT 845 A-138655.1 AGCACCAGCAGGUACUUUAdTdT 1096 AD-68952.1 A-138656.1 CUGGUGCUGAGGGGCUCUAdTdT 846 A-138657.1 UAGAGCCCCUCAGCACCAGdTdT 1097 AD-68953.1 A-138658.1 AGGGGCUCUCCACCUUUCAdTdT 847 A-138659.1 UGAAAGGUGGAGAGCCCCUdTdT 1098 AD-68954.1 A-138660.1 CCUUUCCCAGUUUUUCACUdTdT 848 A-138661.1 AGUGAAAAACUGGGAAAGGdTdT 1099 AD-68955.1 A-138662.1 UUUUUCACUAGAGAAGAGUdTdT 849 A-138663.1 ACUCUUCUCUAGUGAAAAAdTdT 1100 AD-68956.1 A-138664.1 AAGAGUCUGUGAGUCACUUdTdT 850 A-138665.1 AAGUGACUCACAGACUCUUdTdT 1101 AD-68957.1 A-138666.1 AGUCACUUGAGGAGGCGAAdTdT 851 A-138667.1 UUCGCCUCCUCAAGUGACUdTdT 1102 AD-68958.1 A-138668.1 AGGAGGCGAGUCUAGCAGAdTdT 852 A-138669.1 UCUGCUAGACUCGCCUCCUdTdT 1103 AD-68959.1 A-138670.1 AGCAGAUUCUUUCAGAGGUdTdT 853 A-138671.1 ACCUCUGAAAGAAUCUGCUdTdT 1104 AD-68960.1 A-138672.1 UUCAGAGGUGCUAAAGUUUdTdT 854 A-138673.1 AAACUUUAGCACCUCUGAAdTdT 1105 AD-68961.1 A-138674.1 UAAAGUUUCCCAUCUUUGUdTdT 855 A-138675.1 ACAAAGAUGGGAAACUUUAdTdT 1106 AD-68962.1 A-138676.1 UCUUUGUGCAGCUACCUCAdTdT 856 A-138677.1 UGAGGUAGCUGCACAAAGAdTdT 1107 AD-68963.1 A-138678.1 AGCUACCUCCGCAUUGCUAdTdT 857 A-138679.1 UAGCAAUGCGGAGGUAGCUdTdT 1108 AD-68964.1 A-138680.1 UUGCUGUGUAGUGACCCCUdTdT 858 A-138681.1 AGGGGUCACUACACAGCAAdTdT 1109 AD-68965.1 A-138682.1 UGACCCCUGCCUGUGACGUdTdT 859 A-138683.1 ACGUCACAGGCAGGGGUCAdTdT 1110 AD-68966.1 A-138684.1 UGUGACGUGGAGGAUCCCAdTdT 860 A-138685.1 UGGGAUCCUCCACGUCACAdTdT 1111 AD-68967.1 A-138686.1 AGGAUCCCAGCCUCUGAGAdTdT 861 A-138687.1 UCUCAGAGGCUGGGAUCCUdTdT 1112 AD-68968.1 A-138688.1 CUCUGAGCUGAGUUGGUUUdTdT 862 A-138689.1 AAACCAACUCAGCUCAGAGdTdT 1113 AD-68969.1 A-138690.1 UUGGUUUUAUGAAAAGCUAdTdT 863 A-138691.1 UAGCUUUUCAUAAAACCAAdTdT 1114 AD-68970.1 A-138692.1 AAAAGCUAGGAAGCAACCUdTdT 864 A-138693.1 AGGUUGCUUCCUAGCUUUUdTdT 1115 AD-68971.1 A-138694.1 GAAGCAACCUUUCGCCUGUdTdT 865 A-138695.1 ACAGGCGAAAGGUUGCUUCdTdT 1116 AD-68972.1 A-138696.1 UCGCCUGUGCAGCGGUCCAdTdT 866 A-138697.1 UGGACCGCUGCACAGGCGAdTdT 1117 AD-68973.1 A-138698.1 CGGUCCAGCACUUAACUCUdTdT 867 A-138699.1 AGAGUUAAGUGCUGGACCGdTdT 1118 AD-68974.1 A-138700.1 UUAACUCUAAUACAUCAGAdTdT 868 A-138701.1 UCUGAUGUAUUAGAGUUAAdTdT 1119 AD-68975.1 A-138702.1 UACAUCAGCAUGCGUUAAUdTdT 869 A-138703.1 AUUAACGCAUGCUGAUGUAdTdT 1120 AD-68976.1 A-138704.1 CGUUAAUUCAGCUGGUUGAdTdT 870 A-138705.1 UCAACCAGCUGAAUUAACGdTdT 1121 AD-68977.1 A-138706.1 CUGGUUGGGAAAUGACACAdTdT 871 A-138707.1 UGUGUCAUUUCCCAACCAGdTdT 1122 AD-68978.1 A-138708.1 AAUGACACCAGGAAGCCCAdTdT 872 A-138709.1 UGGGCUUCCUGGUGUCAUUdTdT 1123 AD-68979.1 A-138710.1 AAGCCCAGUGCAGAGGGUAdTdT 873 A-138711.1 UACCCUCUGCACUGGGCUUdTdT 1124 AD-68980.1 A-138712.1 AGAGGGUCCCUUACUGACUdTdT 874 A-138713.1 AGUCAGUAAGGGACCCUCUdTdT 1125 AD-68981.1 A-138714.1 UUACUGACUGUUUCGUGGAdTdT 875 A-138715.1 UCCACGAAACAGUCAGUAAdTdT 1126 AD-68982.1 A-138716.1 UUCGUGGCCCUAUUAAUGAdTdT 876 A-138717.1 UCAUUAAUAGGGCCACGAAdTdT 1127 AD-68983.1 A-138718.1 UUAAUGGUCAGACUGUUCAdTdT 877 A-138719.1 UGAACAGUCUGACCAUUAAdTdT 1128 AD-68984.1 A-138720.1 GACUGUUCCAGCAUGAGGUdTdT 878 A-138721.1 ACCUCAUGCUGGAACAGUCdTdT 1129 AD-68985.1 A-138722.1 UGAGGUUCUUAGAAUGACAdTdT 879 A-138723.1 UGUCAUUCUAAGAACCUCAdTdT 1130 AD-68986.1 A-138724.1 UAGAAUGACAGGUGUUUGAdTdT 880 A-138725.1 UCAAACACCUGUCAUUCUAdTdT 1131 AD-68987.1 A-138726.1 UGUUUGGAUGGGUGGGGGAdTdT 881 A-138727.1 UCCCCCACCCAUCCAAACAdTdT 1132 AD-68988.1 A-138728.1 UGGGGGCCUUGUGAUGGGAdTdT 882 A-138729.1 UCCCAUCACAAGGCCCCCAdTdT 1133 AD-68989.1 A-138730.1 UGUGAUGGGGGGUAGGCUAdTdT 883 A-138731.1 UAGCCUACCCCCCAUCACAdTdT 1134 AD-68990.1 A-138732.1 UAGGCUGGCCCAUGUGUGAdTdT 884 A-138733.1 UCACACAUGGGCCAGCCUAdTdT 1135 AD-68991.1 A-138734.1 UGUGUGAUCUUGUGGGGUAdTdT 885 A-138735.1 UACCCCACAAGAUCACACAdTdT 1136 AD-68992.1 A-138736.1 UUGUGGGGUGGAGGGAAGAdTdT 886 A-138737.1 UCUUCCCUCCACCCCACAAdTdT 1137 AD-68993.1 A-138738.1 AGGGAAGAGAAUAGCAUGAdTdT 887 A-138739.1 UCAUGCUAUUCUCUUCCCUdTdT 1138 AD-68994.1 A-138740.1 UAGCAUGAUCCCACUUCCAdTdT 888 A-138741.1 UGGAAGUGGGAUCAUGCUAdTdT 1139 AD-68995.1 A-138742.1 ACUUCCCCAUGCUGUGGGAdTdT 889 A-138743.1 UCCCACAGCAUGGGGAAGUdTdT 1140 AD-68996.1 A-138744.1 CUGUGGGAAGGGGUGCAGUdTdT 890 A-138745.1 ACUGCACCCCUUCCCACAGdTdT 1141 AD-68997.1 A-138746.1 GUGCAGUUCGUCCCCAAGAdTdT 891 A-138747.1 UCUUGGGGACGAACUGCACdTdT 1142 AD-68998.1 A-138748.1 UCCCCAAGAACGACACUGAdTdT 892 A-138749.1 UCAGUGUCGUUCUUGGGGAdTdT 1143 AD-69014.1 A-138750.1 ACACUGCCUGUCAGGUGGUdTdT 893 A-138751.1 ACCACCUGACAGGCAGUGUdTdT 1144 AD-69015.1 A-138752.1 UCAGGUGGUCUGCAAAGAUdTdT 894 A-138753.1 AUCUUUGCAGACCACCUGAdTdT 1145 AD-69016.1 A-138754.1 UGCAAAGAUGAUAACCUUAdTdT 895 A-138755.1 UAAGGUUAUCAUCUUUGCAdTdT 1146 AD-69017.1 A-138756.1 AACCUUGACUACUAAAAACdTdT 896 A-138757.1 GUUUUUAGUAGUCAAGGUUdTdT 1147 AD-69018.1 A-138758.1 UAAAAACGUCUCCAUGGCAdTdT 897 A-138759.1 UGCCAUGGAGACGUUUUUAdTdT 1148 AD-69019.1 A-138760.1 CCAUGGCGGGGGUAACAAAdTdT 898 A-138761.1 UUUGUUACCCCCGCCAUGGdTdT 1149 AD-69020.1 A-138762.1 GGUAACAAGAUGAUAAUCUdTdT 899 A-138763.1 AGAUUAUCAUCUUGUUACCdTdT 1150 AD-69021.1 A-138764.1 UGAUAAUCUACUUAAUUUUdTdT 900 A-138765.1 AAAAUUAAGUAGAUUAUCAdTdT 1151 AD-69022.1 A-138766.1 UUAAUUUUAGAACACCUUUdTdT 901 A-138767.1 AAAGGUGUUCUAAAAUUAAdTdT 1152 AD-69023.1 A-138768.1 ACACCUUUUUCACCUAACUdTdT 902 A-138769.1 AGUUAGGUGAAAAAGGUGUdTdT 1153 AD-69024.1 A-138770.1 CCUAACUAAAAUAAUGUUUdTdT 903 A-138771.1 AAACAUUAUUUUAGUUAGGdTdT 1154 AD-69025.1 A-138772.1 AUAAUGUUUAAAGAGUUUUdTdT 904 A-138773.1 AAAACUCUUUAAACAUUAUdTdT 1155 AD-69026.1 A-138774.1 GAGUUUUGUAUAAAAAUGUdTdT 905 A-138775.1 ACAUUUUUAUACAAAACUCdTdT 1156 AD-69027.1 A-138776.1 AAAAAUGUAAGGAAGCGUUdTdT 906 A-138777.1 AACGCUUCCUUACAUUUUUdTdT 1157 AD-69028.1 A-138778.1 GGAAGCGUUGUUACCUGUUdTdT 907 A-138779.1 AACAGGUAACAACGCUUCCdTdT 1158 AD-69029.1 A-138780.1 UACCUGUUGAAUUUUGUAUdTdT 908 A-138781.1 AUACAAAAUUCAACAGGUAdTdT 1159 AD-69030.1 A-138782.1 UUGUAUUAUGUGAAUCAGUdTdT 909 A-138783.1 ACUGAUUCACAUAAUACAAdTdT 1160 AD-69031.1 A-138784.1 GAAUCAGUGAGAUGUUAGUdTdT 910 A-138785.1 ACUAACAUCUCACUGAUUCdTdT 1161 AD-69032.1 A-138786.1 AUGUUAGUAGAAUAAGCCUdTdT 911 A-138787.1 AGGCUUAUUCUACUAACAUdTdT 1162 AD-69033.1 A-138788.1 AUAAGCCUUAAAAAAAAAAdTdT 912 A-138789.1 UUUUUUUUUUAAGGCUUAUdTdT 1163 AD-69034.1 A-138790.1 AAAAAAAAAAAAAUCGGUUdTdT 913 A-138791.1 AACCGAUUUUUUUUUUUUUdTdT 1164 AD-69035.1 A-138792.1 AAUCGGUUGGGUGCAGUGAdTdT 914 A-138793.1 UCACUGCACCCAACCGAUUdTdT 1165 AD-69036.1 A-138794.1 UGCAGUGGCACACGGCUGUdTdT 915 A-138795.1 ACAGCCGUGUGCCACUGCAdTdT 1166 AD-69037.1 A-138796.1 GGCUGUAAUCCCAGCACUUdTdT 916 A-138797.1 AAGUGCUGGGAUUACAGCCdTdT 1167 AD-69038.1 A-138798.1 CAGCACUUUGGGAGGCCAAdTdT 917 A-138799.1 UUGGCCUCCCAAAGUGCUGdTdT 1168 AD-69039.1 A-138800.1 GAGGCCAAGGUUGGCAGAUdTdT 918 A-138801.1 AUCUGCCAACCUUGGCCUCdTdT 1169 AD-69040.1 A-138802.1 UUGGCAGAUCACCUGAGGUdTdT 919 A-138803.1 ACCUCAGGUGAUCUGCCAAdTdT 1170 AD-69041.1 A-138804.1 CUGAGGUCAGGAGUUCAAAdTdT 920 A-138805.1 UUUGAACUCCUGACCUCAGdTdT 1171 AD-69042.1 A-138806.1 GAGUUCAAGACCAGUCUGAdTdT 921 A-138807.1 UCAGACUGGUCUUGAACUCdTdT 1172 AD-69043.1 A-138808.1 CAGUCUGGCCAACAUAGCAdTdT 922 A-138809.1 UGCUAUGUUGGCCAGACUGdTdT 1173 AD-69044.1 A-138810.1 AACAUAGCAAAACCCUGUAdTdT 923 A-138811.1 UACAGGGUUUUGCUAUGUUdTdT 1174 AD-69045.1 A-138812.1 CCCUGUCUCUACUAAAAAUdTdT 924 A-138813.1 AUUUUUAGUAGAGACAGGGdTdT 1175 AD-69046.1 A-138814.1 CUAAAAAUACAAAAAUUAUdTdT 925 A-138815.1 AUAAUUUUUGUAUUUUUAGdTdT 1176 AD-69047.1 A-138816.1 AAAAUUAUCUGGGCAUGGUdTdT 926 A-138817.1 ACCAUGCCCAGAUAAUUUUdTdT 1177 AD-69048.1 A-138818.1 GGCAUGGUGGUGCAUGCCUdTdT 927 A-138819.1 AGGCAUGCACCACCAUGCCdTdT 1178 AD-69049.1 A-138820.1 CAUGCCUGUAAUCCCAGCUdTdT 928 A-138821.1 AGCUGGGAUUACAGGCAUGdTdT 1179 AD-69050.1 A-138822.1 AAUCCCAGCUAUUCGGAAAdTdT 929 A-138823.1 UUUCCGAAUAGCUGGGAUUdTdT 1180 AD-69051.1 A-138824.1 UUCGGAAGGCUGAGGCAGAdTdT 930 A-138825.1 UCUGCCUCAGCCUUCCGAAdTdT 1181 AD-69052.1 A-138826.1 AGGCAGGAGAAUCACUUGAdTdT 931 A-138827.1 UCAAGUGAUUCUCCUGCCUdTdT 1182 AD-69053.1 A-138828.1 AUCACUUGAACCCAGGAGAdTdT 932 A-138829.1 UCUCCUGGGUUCAAGUGAUdTdT 1183 AD-69054.1 A-138830.1 CAGGAGGCGGAGGUUGCGAdTdT 933 A-138831.1 UCGCAACCUCCGCCUCCUGdTdT 1184 AD-69055.1 A-138832.1 GUUGCGGUGAGCUGAGAUUdTdT 934 A-138833.1 AAUCUCAGCUCACCGCAACdTdT 1185 AD-69056.1 A-138834.1 CUGAGAUUGCACCAUUUCAdTdT 935 A-138835.1 UGAAAUGGUGCAAUCUCAGdTdT 1186 AD-69057.1 A-138836.1 CACCAUUUCAUUCCAGCCUdTdT 936 A-138837.1 AGGCUGGAAUGAAAUGGUGdTdT 1187 AD-69058.1 A-138838.1 CAGCCUGGGCAACAUGAGUdTdT 937 A-138839.1 ACUCAUGUUGCCCAGGCUGdTdT 1188 AD-69059.1 A-138840.1 AACAUGAGUGAAAGUCUGAdTdT 938 A-138841.1 UCAGACUUUCACUCAUGUUdTdT 1189 AD-69060.1 A-138842.1 AGUCUGACUCAAAAAAAAAdTdT 939 A-138843.1 UUUUUUUUUGAGUCAGACUdTdT 1190 AD-69061.1 A-138844.1 AAAAAAAAAAAAUUUAAAAdTdT 940 A-138845.1 UUUUAAAUUUUUUUUUUUUdTdT 1191 AD-69062.1 A-138846.1 UUUAAAAAACAAAAUAAUAdTdT 941 A-138847.1 UAUUAUUUUGUUUUUUAAAdTdT 1192 AD-69063.1 A-138848.1 AAAAUAAUCUAGUGUGCAAdTdT 942 A-138849.1 UUGCACACUAGAUUAUUUUdTdT 1193 AD-69064.1 A-138850.1 GUGUGCAGGGCAUUCACCUdTdT 943 A-138851.1 AGGUGAAUGCCCUGCACACdTdT 1194 AD-69065.1 A-138852.1 CAUUCACCUCAGCCCCCCAdTdT 944 A-138853.1 UGGGGGGCUGAGGUGAAUGdTdT 1195 AD-69066.1 A-138854.1 CCCCCAGGCAGGAGCCAAAdTdT 945 A-138855.1 UUUGGCUCCUGCCUGGGGGdTdT 1196 AD-69067.1 A-138856.1 AGGAGCCAAGCACAGCAGAdTdT 946 A-138857.1 UCUGCUGUGCUUGGCUCCUdTdT 1197 AD-69068.1 A-138858.1 ACAGCAGGAGCUUCCGCCUdTdT 947 A-138859.1 AGGCGGAAGCUCCUGCUGUdTdT 1198 AD-69069.1 A-138860.1 UUCCGCCUCCUCUCCACUAdTdT 948 A-138861.1 UAGUGGAGAGGAGGCGGAAdTdT 1199 AD-69070.1 A-138862.1 UCCACUGGAGCACACAACUdTdT 949 A-138863.1 AGUUGUGUGCUCCAGUGGAdTdT 1200 AD-69071.1 A-138864.1 ACACAACUUGAACCUGGCUdTdT 950 A-138865.1 AGCCAGGUUCAAGUUGUGUdTdT 1201 AD-69072.1 A-138866.1 AACCUGGCUUAUUUUCUGAdTdT 951 A-138867.1 UCAGAAAAUAAGCCAGGUUdTdT 1202 AD-69073.1 A-138868.1 UUCUGCAGGGACCAGCCCAdTdT 952 A-138869.1 UGGGCUGGUCCCUGCAGAAdTdT 1203 AD-69074.1 A-138870.1 CCAGCCCCACAUGGUCAGUdTdT 953 A-138871.1 ACUGACCAUGUGGGGCUGGdTdT 1204 AD-69076.1 A-138874.1 UUUCUCCCCAUGUGUGGCAdTdT 954 A-138875.1 UGCCACACAUGGGGAGAAAdTdT 1205 AD-69077.1 A-138878.1 AGAGAGUGUAGAAAUAAAGdTdT 955 A-138879.1 CUUUAUUUCUACACUCUCUdTdT 1206

TABLE 6 PNPLA3 Single Dose Screen in Hep3B Cells Data are expressed as percent message remaining relative to AD-1955, a non-targeting control duplex. Duplex Name 20 nM_AVG 20 nM_STDEV AD-68792.1 106.53 9.20 AD-68793.1 90.00 15.49 AD-68794.1 55.08 11.00 AD-68795.1 77.11 20.01 AD-68796.1 71.27 7.67 AD-68797.1 53.86 1.23 AD-68798.1 76.58 29.01 AD-68799.1 61.71 33.05 AD-68800.1 84.27 23.89 AD-68801.1 58.51 23.74 AD-68802.1 48.71 3.47 AD-68803.1 52.69 8.91 AD-68804.1 56.10 9.15 AD-68805.1 56.10 29.42 AD-68806.1 52.09 4.59 AD-68807.1 69.70 8.99 AD-68808.1 83.88 7.42 AD-68809.1 67.95 17.68 AD-68810.1 52.56 22.52 AD-68811.1 73.72 12.31 AD-68812.1 70.61 22.53 AD-68813.1 63.84 17.87 AD-68814.1 56.57 4.47 AD-68815.1 50.13 8.52 AD-68816.1 91.97 18.35 AD-68817.1 49.93 3.88 AD-68818.1 74.08 23.36 AD-68819.1 74.87 26.63 AD-68820.1 59.47 18.45 AD-68821.1 81.26 37.48 AD-68822.1 63.53 8.85 AD-68823.1 49.54 9.87 AD-68824.1 87.65 12.09 AD-68825.1 107.35 28.04 AD-68826.1 100.30 41.14 AD-68827.1 62.87 13.83 AD-68828.1 63.50 18.27 AD-68829.1 40.09 7.84 AD-68830.1 32.34 4.08 AD-68831.1 46.76 7.68 AD-68832.1 78.43 16.54 AD-68833.1 125.50 3.95 AD-68834.1 112.62 6.58 AD-68835.1 97.95 2.75 AD-68836.1 117.74 52.61 AD-68837.1 40.88 4.78 AD-68838.1 91.56 20.60 AD-68839.1 59.94 8.72 AD-68840.1 79.60 5.47 AD-68841.1 39.27 7.63 AD-68842.1 88.01 18.52 AD-68843.1 56.54 5.00 AD-68844.1 51.39 10.45 AD-68845.1 59.74 4.73 AD-68846.1 54.54 14.99 AD-68847.1 94.59 4.92 AD-68848.1 92.93 14.62 AD-68849.1 74.04 7.30 AD-68850.1 110.43 16.00 AD-68851.1 61.74 5.05 AD-68852.1 63.66 21.55 AD-68853.1 49.87 6.96 AD-68854.1 47.59 6.65 AD-68855.1 73.32 11.72 AD-68856.1 106.96 18.30 AD-68857.1 123.97 37.64 AD-68858.1 60.42 4.02 AD-68859.1 81.29 14.80 AD-68860.1 68.06 17.18 AD-68861.1 89.36 8.04 AD-68862.1 62.20 19.06 AD-68863.1 78.73 13.90 AD-68864.1 71.54 10.06 AD-68865.1 79.83 18.10 AD-68866.1 90.56 9.37 AD-68867.1 76.38 25.29 AD-68868.1 106.98 9.34 AD-68869.1 80.37 23.99 AD-68870.1 62.13 19.67 AD-68871.1 82.72 12.73 AD-68872.1 78.95 8.19 AD-68873.1 71.57 3.92 AD-68874.1 118.98 25.63 AD-68875.1 82.64 10.49 AD-68876.1 106.02 17.93 AD-68877.1 47.83 10.91 AD-68878.1 68.77 9.18 AD-68879.1 92.72 13.76 AD-68880.1 121.48 13.92 AD-68881.1 99.48 5.55 AD-68882.1 90.81 8.65 AD-68883.1 88.48 16.79 AD-68884.1 126.40 27.97 AD-68885.1 79.31 13.00 AD-68905.1 59.11 11.11 AD-68906.1 62.09 23.14 AD-68907.1 91.47 18.05 AD-68908.1 69.14 6.98 AD-68909.1 57.61 0.00 AD-68910.1 53.43 6.58 AD-68911.1 49.21 4.14 AD-68912.1 55.29 11.49 AD-68913.1 60.30 3.64 AD-68914.1 64.75 6.02 AD-68915.1 77.72 5.80 AD-68916.1 51.18 6.74 AD-68917.1 61.47 5.86 AD-68918.1 63.11 5.98 AD-68919.1 58.34 10.77 AD-68920.1 50.34 15.08 AD-68921.1 82.27 16.34 AD-68922.1 76.90 14.57 AD-68923.1 73.35 4.56 AD-68924.1 54.86 10.39 AD-68925.1 79.75 12.87 AD-68926.1 67.63 6.30 AD-68927.1 70.30 11.39 AD-68928.1 71.51 12.69 AD-68929.1 66.30 18.72 AD-68930.1 71.14 21.97 AD-68931.1 71.05 8.92 AD-68932.1 77.92 4.34 AD-68933.1 101.43 16.21 AD-68934.1 53.20 9.90 AD-68935.1 99.51 9.41 AD-68936.1 49.46 8.03 AD-68937.1 57.51 13.53 AD-68938.1 88.20 15.56 AD-68939.1 74.32 14.17 AD-68940.1 77.38 17.70 AD-68941.1 76.90 11.02 AD-68942.1 86.39 14.95 AD-68943.1 110.51 36.72 AD-68944.1 66.71 10.77 AD-68945.1 70.73 19.44

Example 3. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

siRNA Design

A set of iRNAs targeting the human PNPLA3 (human: NCBI refseqID NM_025225; NCBI GeneID: 80339), as well as toxicology-species PNPLA3 orthologs (cynomolgus monkey: XM_005567051; mouse: NM_054088; rat: XM_006242109) were designed using custom R and Python scripts. The human PNPLA3 REFSEQ mRNA has a length of 2805 bases. The rationale and method for the set of iRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 174 through position 2805 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey, mouse, and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8, 1.2 and 1 for seed mismatches, cleavage site, and other positions up through antisense position 19, respectively. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=3.0 and predicted efficacy was >=70% knockdown of the PNPLA3 transcript.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 7. A detailed list of the modified PNPLA3 sense and antisense strand sequences is shown in Table 8. Table 9 provides the mRNA target sequences of the modified PNPLA3 agents provided in Table 8

In Vitro Screening

Cell Culture and Transfections

Hep3b cells, mouse and cynomolgus monkey primary hepatocytes were transfected, independently, by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of iRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of EMEM containing about 5×10³ Hep3b cells, or 40 μl of William's media containing about 5×10³ primary mouse hepatocytes or primary cynomolgus monkey hepatocytes were then added to the iRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Two single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations and dose response experiments were performed over a range of doses from 10 nM to 36 fM final duplex concentration over 8, 6-fold dilutions.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905), 0.5 μl PNPLA3 probe and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Hep3b qPCR was probed with GAPDH TaqMan Probe (Hs99999905) and PNPLA3 probe (Hs00228747_m1). Mouse primary hepatocytes qPCR was probed with Mouse GAPDH TaqMan Probe (Mm03302249_g1) and Mouse PNPLA3 Taqman Probe (Mm00504420_m1). Cynomolgus monkey primary hepatocytes qPCR was probed with custom Cynomolgus GAPDH probe and custom Cynomolgus PNPLA3 probe (5′-AGCGGGGUCUGAAGUCAU-3′(SEQ ID NO: 1207)). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, a non-targeting control iRNA, or mock transfected cells. The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 1208) cuuAcGcuGAGuAcuucGAdTsdT; ANTISENSE: (SEQ ID NO: 1209) UCGAAGuACUcAGCGuAAGdTsdT.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Dual-Glo® Luciferase constructs were generated in the psiCHECK2 plasmid and contained approximately 2.8 kb (human) PNPLA3 sequences (SEQ ID NO:18). Dual-luciferase plasmids were co-transfected with siRNA into 15×10³ cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 96 well plate, 0.2 μl of Lipofectamine were added to 10 ng of plasmid vector and iRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 80 μl of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Two single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations and dose response experiments were performed over a range of doses from 10 nM to 36 fM final duplex concentration over 8, 6-fold dilutions.

Dual-Glo® Luciferase Assay

Fifty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to PNPLA3 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 75 μl of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture was incubated at room temperature for 30 minutes before luminescence (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 75 μl of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent, quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. iRNA activity was determined by normalizing the Renilla (PNPLA3) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with iRNA or were treated with a non-targeting iRNA. All transfections were done in triplicate.

Table 10 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Hep3B cells transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 11 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Cynomolgus monkey primary hepatocytes transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells. Table 12 shows the dose response in primary Cynomolgus monkey hepatocytes transfected with the indicated modified RNAi agents. The indicated IC₅₀ values represent the IC₅₀ values relative to untreated cells.

Table 13 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in mouse primary hepatocytes transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 14 shows the dose response in primary mouse hepatocytes transfected with the indicated modified RNAi agents. The indicated IC₅₀ values represent the IC₅₀ values relative to untreated cells.

Table 15 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Cos 7 cells transfected with the indicated modified PNPLA3 RNAi agents. Data are expressed as percent of mRNA remaining relative to negative control.

Table 16 shows the dose response in Cos 7 cells transfected with the indicated modified RNAi agents. The indicated IC₅₀ values represent the IC₅₀ values relative to untreated cells.

TABLE 7 PNPLA3 Unmodified Sequences Start Start in in Sense Sense SEQ SEQ Antisense Antisense SEQ SEQ Duplex Oligo Sequence ID ID Oligo Sequence ID ID Name Name 5′ to 3′ NO NO: 1 Name 5′ to 3′ NO NO: 1 Range AD-67524.1 A-135231.1 GGCU 1210 219 A-135232.1 UUGG 1300 217 217-239 UCCU UAGA GGGC AGCC UUCU CAGG ACCA AAGC A CGC AD-67611.1 A-135409.1 UUGU 1211 388 A-135410.1 UACU 1301 386 386-408 GCGG CCUG AAGG GCCU CCAG UCCG GAGU CACA A AGA AD-67601.1 A-135389.1 AAGG 1212 396 A-135390.1 AAUG 1302 394 394-416 CCAG UUCC GAGU GACU CGGA CCUG ACAU GCCU U UCC AD-67579.1 A-135341.1 AGGC 1213 397 A-135342.1 UAAU 1303 395 395-417 CAGG GUUC AGUC CGAC GGAA UCCU CAUU GGCC A UUC AD-67588.1 A-135361.1 AACG 1214 549 A-135362.1 AAAG 1304 547 547-569 UUCU UCAG GGUG ACAC UCUG CAGA ACUU ACGU U UUU AD-67602.1 A-135391.1 CUGA 1215 562 A-135392.1 UGUC 1305 560 560-582 CUUU UUUG CGGU GACC CCAA GAAA AGAC GUCA A GAC AD-67570.1 A-135323.1 UCGG 1216 569 A-135324.1 ACGA 1306 567 567-589 UCCA CUUC AAGA GUCU CGAA UUGG GUCG ACCG U AAA AD-67553.1 A-135289.1 CGGU 1217 570 A-135290.1 UACG 1307 568 568-590 CCAA ACUU AGAC CGUC GAAG UUUG UCGU GACC A GAA AD-67612.1 A-135411.1 GACG 1218 579 A-135412.1 UAAG 1308 577 577-599 AAGU GCAU CGUG CCAC GAUG GACU CCUU UCGU A CUU AD-67525.1 A-135233.1 CUUG 1219 596 A-135234.1 AUGA 1309 594 594-616 GUAU AGCA GUUC GGAA CUGC CAUA UUCA CCAA U GGC AD-67526.1 A-135235.1 GGCC 1220 630 A-135236.1 UAAG 1310 628 628-650 UUAU GAAG CCCU GAGG CCUU GAUA CCUU AGGC A CAC AD-67592.1 A-135371.1 AGGA 1221 674 A-135372.1 UGUA 1311 672 672-694 GUGA CGUU GUGA GUCA CAAC CUCA GUAC CUCC A UCC AD-67578.1 A-135339.1 GUGA 1222 678 A-135340.1 UAAG 1312 676 676-698 GUGA GGUA CAAC CGUU GUAC GUCA CCUU CUCA A CUC AD-67555.1 A-135293.1 UGAU 1223 701 A-135294.1 UUGA 1313 699 699-721 GCCA UGGU AAAC UGUU AACC UUGG AUCA CAUC A AAU AD-67577.1 A-135337.1 CGAC 1224 746 A-135338.1 UUGA 1314 744 744-766 AUCU CUUU GCCC AGGG UAAA CAGA GUCA UGUC A GUA AD-67594.1 A-135375.1 ACGA 1225 771 A-135376.1 UUCC 1315 769 769-791 ACUU ACAU UCUU GAAG CAUG AAAG UGGA UUCG A UGG AD-67568.1 A-135319.1 GCAC 1226 817 A-135320.1 UAAG 1316 815 815-837 AGGG GUAG AACC AGGU UCUA UCCC CCUU UGUG A CAG AD-67550.1 A-135283.1 UGCU 1227 871 A-135284.1 UAAG 1317 869 869-891 GGGA GCAU GAGA AUCU UAUG CUCC CCUU CAGC A ACC AD-67586.1 A-135357.1 UGGG 1228 874 A-135358.1 UUCG 1318 872 872-894 AGAG AAGG AUAU CAUA GCCU UCUC UCGA UCCC A AGC AD-67576.1 A-135335.1 AGAG 1229 878 A-135336.1 UAUC 1319 876 876-898 AUAU CUCG GCCU AAGG UCGA CAUA GGAU UCUC A UCC AD-67563.1 A-135309.1 AUAU 1230 882 A-135310.1 UAAA 1320 880 880-902 GCCU UAUC UCGA CUCG GGAU AAGG AUUU CAUA A UCU AD-67552.1 A-135287.1 UGCC 1231 885 A-135288.1 AUCC 1321 883 883-905 UUCG AAAU AGGA AUCC UAUU UCGA UGGA AGGC U AUA AD-67608.1 A-135403.1 AUUC 1232 908 A-135404.1 UUCU 1322 906 906-928 AGGU CUUC UCUU CAAG GGAA AACC GAGA UGAA A UGC AD-67593.1 A-135373.1 CAUC 1233 964 A-135374.1 UAUC 1323 962 962-984 CUCA CAUC GAAG CCUU GGAU CUGA GGAU GGAU A GAC AD-67609.1 A-135405.1 CCUG 1234 1100 A-135406.1 AUGC 1324 1098 1098-1120 CCCU UCUC GGGA AUCC UGAG CAGG AGCA GCAG U GAU AD-67597.1 A-135381.1 GACA 1235 1173 A-135382.1 UCUC 1325 1171 1171-1193 AAGG AUGU UGGA AUCC UACA ACCU UGAG UUGU A CUU AD-67587.1 A-135359.1 AAAG 1236 1176 A-135360.1 UUUG 1326 1174 1174-1196 GUGG CUCA AUAC UGUA AUGA UCCA GCAA CCUU A UGU AD-67559.1 A-135301.1 GUGG 1237 1180 A-135302.1 AAAU 1327 1178 1178-1200 AUAC CUUG AUGA CUCA GCAA UGUA GAUU UCCA U CCU AD-67561.1 A-135305.1 UGGA 1238 1181 A-135306.1 UAAA 1328 1179 1179-1201 UACA UCUU UGAG GCUC CAAG AUGU AUUU AUCC A ACC AD-67551.1 A-135285.1 AUAC 1239 1184 A-135286.1 UUGC 1329 1182 1182-1204 AUGA AAAU GCAA CUUG GAUU CUCA UGCA UGUA A UCC AD-67591.1 A-135369.1 AGCA 1240 1191 A-135370.1 UAGC 1330 1189 1189-1211 AGAU AAGU UUGC UGCA AACU AAUC UGCU UUGC A UCA AD-67583.1 A-135351.1 CAAG 1241 1193 A-135352.1 UGUA 1331 1191 1191-1213 AUUU GCAA GCAA GUUG CUUG CAAA CUAC UCUU A GCU AD-67585.1 A-135355.1 UGCA 1242 1200 A-135356.1 UCUA 1332 1198 1198-1220 ACUU AUGG GCUA GUAG CCCA CAAG UUAG UUGC A AAA AD-67589.1 A-135363.1 AACU 1243 1203 A-135364.1 UAUC 1333 1201 1201-1223 UGCU CUAA ACCC UGGG AUUA UAGC GGAU AAGU A UGC AD-67595.1 A-135377.1 GCCA 1244 1266 A-135378.1 UCUC 1334 1264 1264-1286 UUGC UGGA GAUU CAAU GUCC CGCA AGAG AUGG A CAG AD-67580.1 A-135343.1 GAUU 1245 1274 A-135344.1 UUCA 1335 1272 1272-1294 GUCC CCAG AGAG UCUC ACUG UGGA GUGA CAAU A CGC AD-67573.1 A-135329.1 UGGU 1246 1288 A-135330.1 UAUC 1336 1286 1286-1308 GACA UGGA UGGC AGCC UUCC AUGU AGAU CACC A AGU AD-67600.1 A-135387.1 CCAG 1247 1302 A-135388.1 UACA 1337 1300 1300-1322 AUAU UCGU GCCC CGGG GACG CAUA AUGU UCUG A GAA AD-67603.1 A-135393.1 GUGG 1248 1325 A-135394.1 UAGG 1338 1323 1323-1345 UUGC UCAC AGUG CCAC GGUG UGCA ACCU ACCA A CAG AD-67598.1 A-135383.1 AGGU 1249 1389 A-135384.1 UCUC 1339 1387 1387-1409 CCCA ACUG AAUG GCAU CCAG UUGG UGAG GACC A UGG AD-67564.1 A-135311.1 UCAC 1250 1621 A-135312.1 UAGA 1340 1619 1619-1641 UUGA CUCG GGAG CCUC GCGA CUCA GUCU AGUG A ACU AD-67574.1 A-135331.1 AGUC 1251 1636 A-135332.1 UCUG 1341 1634 1634-1656 UAGC AAAG AGAU AAUC UCUU UGCU UCAG AGAC A UCG AD-67590.1 A-135365.1 AUUC 1252 1646 A-135366.1 UUUU 1342 1644 1644-1666 UUUC AGCA AGAG CCUC GUGC UGAA UAAA AGAA A UCU AD-67572.1 A-135327.1 UUCU 1253 1647 A-135328.1 ACUU 1343 1645 1645-1667 UUCA UAGC GAGG ACCU UGCU CUGA AAAG AAGA U AUC AD-67582.1 A-135349.1 GUGC 1254 1658 A-135350.1 AAAG 1344 1656 1656-1678 UAAA AUGG GUUU GAAA CCCA CUUU UCUU AGCA U CCU AD-67607.1 A-135401.1 UCCC 1255 1669 A-135402.1 UGUA 1345 1667 1667-1689 AUCU GCUG UUGU CACA GCAG AAGA CUAC UGGG A AAA AD-67571.1 A-135325.1 CUGC 1256 1713 A-135326.1 UAUC 1346 1711 1711-1733 CUGU CUCC GACG ACGU UGGA CACA GGAU GGCA A GGG AD-67599.1 A-135385.1 UGUG 1257 1718 A-135386.1 UCUG 1347 1716 1716-1738 ACGU GGAU GGAG CCUC GAUC CACG CCAG UCAC A AGG AD-67554.1 A-135291.1 UCUG 1258 1740 A-135292.1 AUAA 1348 1738 1738-1760 AGCU AACC GAGU AACU UGGU CAGC UUUA UCAG U AGG AD-67549.1 A-135281.1 AGUU 1259 1749 A-135282.1 UAGC 1349 1747 1747-1769 GGUU UUUU UUAU CAUA GAAA AAAC AGCU CAAC A UCA AD-67567.1 A-135317.1 UUGG 1260 1751 A-135318.1 UCUA 1350 1749 1749-1771 UUUU GCUU AUGA UUCA AAAG UAAA CUAG ACCA A ACU AD-67558.1 A-135299.1 GGUU 1261 1753 A-135300.1 UUCC 1351 1751 1751-1773 UUAU UAGC GAAA UUUU AGCU CAUA AGGA AAAC A CAA AD-67569.1 A-135321.1 GUUU 1262 1754 A-135322.1 UUUC 1352 1752 1752-1774 UAUG CUAG AAAA CUUU GCUA UCAU GGAA AAAA A CCA AD-67548.1 A-135279.1 UUUU 1263 1755 A-135280.1 UCUU 1353 1753 1753-1775 AUGA CCUA AAAG GCUU CUAG UUCA GAAG UAAA A ACC AD-67566.1 A-135315.1 UAUG 1264 1758 A-135316.1 UUUG 1354 1756 1756-1778 AAAA CUUC GCUA CUAG GGAA CUUU GCAA UCAU A AAA AD-67613.1 A-135413.1 CGUU 1265 1827 A-135414.1 UCCC 1355 1825 1825-1847 AAUU AACC CAGC AGCU UGGU GAAU UGGG UAAC A GCA AD-67610.1 A-135407.1 GUUA 1266 1828 A-135408.1 UUCC 1356 1826 1826-1848 AUUC CAAC AGCU CAGC GGUU UGAA GGGA UUAA A CGC AD-67556.1 A-135295.1 AGCU 1267 1836 A-135296.1 UGUG 1357 1834 1834-1856 GGUU UCAU GGGA UUCC AAUG CAAC ACAC CAGC A UGA AD-67581.1 A-135345.1 CCUA 1268 1900 A-135346.1 AACA 1358 1898 1898-1920 UUAA GUCU UGGU GACC CAGA AUUA CUGU AUAG U GGC AD-67560.1 A-135303.1 CUAU 1269 1901 A-135304.1 UAAC 1359 1899 1899-1921 UAAU AGUC GGUC UGAC AGAC CAUU UGUU AAUA A GGG AD-67596.1 A-135379.1 GCUG 1270 1984 A-135380.1 UAAG 1360 1982 1982-2004 GCCC AUCA AUGU CACA GUGA UGGG UCUU CCAG A CCU AD-67557.1 A-135297.1 UGGC 1271 1986 A-135298.1 UACA 1361 1984 1984-2006 CCAU AGAU GUGU CACA GAUC CAUG UUGU GGCC A AGC AD-67584.1 A-135353.1 CCUA 1272 2190 A-135354.1 UUAA 1362 2188 2188-2210 ACUA ACAU AAAU UAUU AAUG UUAG UUUA UUAG A GUG AD-67575.1 A-135333.1 UUAC 1273 2243 A-135334.1 AAUA 1363 2241 2241-2263 CUGU CAAA UGAA AUUC UUUU AACA GUAU GGUA U ACA AD-67605.1 A-135397.1 ACCU 1274 2245 A-135398.1 AUAA 1364 2243 2243-2265 GUUG UACA AAUU AAAU UUGU UCAA AUUA CAGG U UAA AD-67562.1 A-135307.1 UGUA 1275 2258 A-135308.1 UUCA 1365 2256 2256-2278 UUAU CUGA GUGA UUCA AUCA CAUA GUGA AUAC A AAA AD-67606.1 A-135399.1 UAUG 1276 2263 A-135400.1 AACA 1366 2261 2261-2283 UGAA UCUC UCAG ACUG UGAG AUUC AUGU ACAU U AAU AD-67604.1 A-135395.1 GAUG 1277 2278 A-135396.1 AAGG 1367 2276 2276-2298 UUAG CUUA UAGA UUCU AUAA ACUA GCCU ACAU U CUC AD-67565.1 A-135313.1 AUGU 1278 2279 A-135314.1 UAAG 1368 2277 2277-2299 UAGU GCUU AGAA AUUC UAAG UACU CCUU AACA A UCU AD-67529.1 A-135241.1 UAUA 1279 250 A-135242.1 UCCA 1369 248 248-270 AUGG UGAG AGAU GAUC CCUC UCCA AUGG UUAU A ACG AD-67533.1 A-135249.1 GUGU 1280 443 A-135250.1 UUUG 1370 441 441-463 CUGA GAAU GUUC GGAA CAUU CUCA CCAA GACA A CCA AD-67537.1 A-135257.1 AGUC 1281 469 A-135258.1 UACA 1371 467 467-489 GUGG CCAG AUGC GGCA CCUG UCCA GUGU CGAC A UUC AD-67546.1 A-135275.1 UGCU 1282 770 A-135276.1 UUCC 1372 768 768-790 AUCA AGGU AGGG ACCC UACC UUGA UGGA UAGC A ACA AD-67547.1 A-135277.1 UCCC 1283 1163 A-135278.1 UAUU 1373 1161 1161-1183 AGGU CGGG UUGU CACA GCCC AACC GAAU UGGG A AUG AD-67543.1 A-135269.1 CCAG 1284 1165 A-135270.1 UUCA 1374 1163 1163-1185 GUUU UUCG GUGC GGCA CCGA CAAA AUGA CCUG A GGA AD-67541.1 A-135265.1 UGGA 1285 3032 A-135266.1 UAUC 1375 3030 3030-3052 GCAA UAGA CAGU CACU GUCU GUUG AGAU CUCC A AGA AD-67535.1 A-135253.1 CUUU 1286 3106 A-135254.1 UUUC 1376 3104 3104-3126 UGGA CUAG GGCA CUGC GCUA CUCC GGAA AAAA A GUA AD-67530.1 A-135243.1 AAGA 1287 3226 A-135244.1 UAAA 1377 3224 3224-3246 CAAU CACC GAUU AAAU UGGU CAUU GUUU GUCU A UUG AD-67542.1 A-135267.1 GACA 1288 3228 A-135268.1 UCUA 1378 3226 3226-3248 AUGA AACA UUUG CCAA GUGU AUCA UUAG UUGU A CUU AD-67528.1 A-135239.1 CAAU 1289 3230 A-135240.1 UUUC 1379 3228 3228-3250 GAUU UAAA UGGU CACC GUUU AAAU AGAA CAUU A GUC AD-67527.1 A-135237.1 UGCC 1290 3447 A-135238.1 AAAG 1380 3445 3445-3467 AGAU UAAU AACU AAGU UAUU UAUC ACUU UGGC U AGG AD-67544.1 A-135271.1 ACAC 1291 3473 A-135272.1 AUUA 1381 3471 3471-3493 CUUU GUAA GGCU GAGC CUUA CAAA CUAA GGUG U UCC AD-67532.1 A-135247.1 CUGG 1292 3629 A-135248.1 UAUA 1382 3627 3627-3649 CUCC CAAA AAAU GAUU CUUU UGGA GUAU GCCA A GUG AD-67534.1 A-135251.1 UGGC 1293 3630 A-135252.1 UUAU 1383 3628 3628-3650 UCCA ACAA AAUC AGAU UUUG UUGG UAUA AGCC A AGU AD-67538.1 A-135259.1 CCAA 1294 3635 A-135260.1 UAUG 1384 3633 3633-3655 AUCU ACUA UUGU UACA AUAG AAGA UCAU UUUG A GAG AD-67545.1 A-135273.1 AGAG 1295 3986 A-135274.1 UAGC 1385 3984 3984-4006 ACAA CUAG AGUG ACAC UCUA UUUG GGCU UCUC A UAG AD-67539.1 A-135261.1 AAGU 1296 3993 A-135262.1 UUCU 1386 3991 3991-4013 GUCU GUGU AGGC AGCC UACA UAGA CAGA CACU A UUG AD-67540.1 A-135263.1 AGAA 1297 4283 A-135264.1 UAAA 1387 4281 4281-4303 ACUU GCAA CUGC GGCA CUUG GAAG CUUU UUUC A UAC AD-67531.1 A-135245.1 GAAG 1298 4540 A-135246.1 UGUG 1388 4538 4538-4560 GAUU UAUC GAAU CAUU GGAU CAAU ACAC CCUU A CUG AD-67536.1 A-135255.1 GGAU 1299 4543 A-135256.1 UUUG 1389 4541 4541-4563 UGAA GUGU UGGA AUCC UACA AUUC CCAA AAUC A CUU

TABLE 8 PNPLA3 Modified Sequences Anti- Sense sense Se- Anti- Se- Sense quence SEQ sense quence SEQ  Duplex Oligo 5′ to ID Oligo 5′ to ID Name Name 3′ NO Name 3′ NO AD- A- gsgs 1390 A- usUf 1480 67524.1 135231.1 cuuc 135232.1 sggu CfuG AfgA fGfG fAfg fcuu cccA cuac fgGf caaL aagc 96 csgs c AD- A- usus 1391 A- usAf 1481 67611.1 135409.1 gugc 135410.1 scuc GfgA CfuG fAfG fGfc fgcc cuuC agga fcGf guaL caca 96 asgs a AD- A- asas 1392 A- asAf 1482 67601.1 135389.1 ggcc 135390.1 sugu AfgG UfcC fAfG fGfa fucg cucC gaac fuGf auuL gccu 96 uscs c AD- A- asgs 1393 A- usAf 1483 67579.1 135341.1 gcca 135342.1 saug GfgA UfuC fGfU fCfg fcgg acuC aaca fcUf uuaL ggcc 96 usus c AD- A- asas 1394 A- asAf 1484 67588.1 135361.1 cguu 135362.1 sagu CfuG CfaG fGfU fAfc fguc accA ugac fgAf uuuL acgu 96 usus u AD- A- csus 1395 A- usGf 1485 67602.1 135391.1 gacu 135392.1 sucu UfuC UfuG fGfG fGfa fucc ccgA aaag faAf acaL guca 96 gsas c AD- A- uscs 1396 A- asCf 1486 67570.1 135323.1 gguc 135324.1 sgac CfaA UfuC fAfG fGfu facg cuuU aagu fgGf cguL accg 96 asas a AD- A- csgs 1397 A- usAf 1487 67553.1 135289.1 gucc 135290.1 scga AfaA CfuU fGfA fCfg fcga ucuU aguc fuGf guaL gacc 96 gsas a AD- A- gsas 1398 A- usAf 1488 67612.1 135411.1 cgaa 135412.1 sagg GfuC CfaU fGfU fCfc fgga acgA ugcc fcUf uuaL ucgu 96 csus u AD- A- csus 1399 A- asUf 1489 67525.1 135233.1 uggu 135234.1 sgaa AfuG GfcA fUfU fGfg fccu aacA gcuu fuAf cauL ccaa 96 gsgs c AD- A- gsgs 1400 A- usAf 1490 67526.1 135235.1 ccuu 135236.1 sagg AfuC AfaG fCfC fGfa fucc gggA uucc fuAf uuaL aggc 96 csas c AD- A- asgs 1401 A- usGf 1491 67592.1 135371.1 gagu 135372.1 suac GfaG GfuU fUfG fGfu faca cacU acgu fcAf acaL cucc 96 uscs c AD- A- gsus 1402 A- usAf 1492 67578.1 135339.1 gagu 135340.1 sagg GfaC GfuA fAfA fCfg fcgu uugU accc fcAf uuaL cuca 96 csus c AD- A- usgs 1403 A- usUf 1493 67555.1 135293.1 augc 135294.1 sgau CfaA GfgU fAfA fUfg fcaa uuuU ccau fgGf caaL cauc 96 asas u AD- A- csgs 1404 A- usUf 1494 67577.1 135337.1 acau 135338.1 sgac CfuG UfuU fCfC fAfg fcua ggcA aagu fgAf caaL uguc 96 gsus a AD- A- ascs 1405 A- usUf 1495 67594.1 135375.1 gaac 135376.1 scca UfuU CfaU fCfU fGfa fuca agaA ugug faGf gaaL uucg 96 usgs g AD- A- gscs 1406 A- usAf 1496 67568.1 135319.1 acag 135320.1 sagg GfgA UfaG fAfC fAfg fcuc guuC uacc fcCf uuaL ugug 96 csas g AD- A- usgs 1407 A- usAf 1497 67550.1 135283.1 cugg 135284.1 sagg GfaG CfaU fAfG fAfu faua cucU ugcc fcCf uuaL cagc 96 ascs c AD- A- usgs 1408 A- usUf 1498 67586.1 135357.1 ggag 135358.1 scga AfgA AfgG fUfA fCfa fugc uauC cuuc fuCf gapL uccc 96 asgs c AD- A- asgs 1409 A- usAf 1499 67576.1 135335.1 agau 135336.1 succ AfuG UfcG fCfC fAfa fuuc ggcA gagg fuAf auaL ucuc 96 uscs c AD- A- asus 1410 A- usAf 1500 67563.1 135309.1 augc 135310.1 saau CfuU AfuC fCfG fCfu fagg cgaA auau fgGf uuaL caua 96 uscs u AD- A- usgs 1411 A- asUf 1501 67552.1 135287.1 ccuu 135288.1 scca CfgA AfaU fGfG fAfu faua ccuC uuug fgAf gauL aggc 96 asus a AD- A- asus 1412 A- usUf 1502 67608.1 135403.1 ucag 135404.1 scuc GfuU UfuC fCfU fCfa fugg agaA aaga fcCf gapL ugaa 96 usgs c AD- A- csas 1413 A- usAf 1503 67593.1 135373.1 uccu 135374.1 succ CfaG AfuC fAfA fCfc fggg uucU augg fgAf auaL ggau 96 gsas c AD- A- cscs 1414 A- asUf 1504 67609.1 135405.1 ugcc 135406.1 sgcu CfuG CfuC fGfG fAfu faug cccA agag fgGf cauL gcag 96 gsas u AD- A- gsas 1415 A- usCf 1505 67597.1 135381.1 caaa 135382.1 suca GfgU UfgU fGfG fAfu faua ccaC caug fcUf agaL uugu 96 csus u AD- A- asas 1416 A- usUf 1506 67587.1 135359.1 aggu 135360.1 sugc GfgA UfcA fUfA fUfg fcau uauC gagc fcAf aaaL ccuu 96 usgs u AD- A- gsus 1417 A- asAf 1507 67559.1 135301.1 ggau 135302.1 sauc AfcA UfuG fUfG fCfu fagc cauG aaga fuAf uuuL ucca 96 cscs u AD- A- usgs 1418 A- usAf 1508 67561.1 135305.1 gaua 135306.1 saau CfaU CfuU fGfA fGfc fgca ucaU agau fgUf uuaL aucc 96 ascs c AD- A- asus 1419 A- usUf 1509 67551.1 135285.1 acau 135286.1 sgca GfaG AfaU fCfA fCfu faga ugcU uuug fcAf caaL ugua 96 uscs c AD- A- asgs 1420 A- usAf 1510 67591.1 135369.1 caag 135370.1 sgca AfuU AfgU fUfG fUfg fcaa caaA cuug fuCf cuaL uugc 96 uscs a AD- A- csas 1421 A- usGf 1511 67583.1 135351.1 agau 135352.1 suag UfuG CfaA fCfA fGfu facu ugcA ugcu faAf acaL ucuu 96 gscs u AD- A- usgs 1422 A- usCf 1512 67585.1 135355.1 caac 135356.1 suaa UfuG UfgG fCfU fGfu facc agcA cauu faGf agaL uugc 96 asas a AD- A- asas 1423 A- usAf 1513 67589.1 135363.1 cuug 135364.1 succ CfuA UfaA fCfC fUfg fcau gguA uagg fgCf auaL aagu 96 usgs c AD- A- gscs 1424 A- usCf 1514 67595.1 135377.1 cauu 135378.1 sucu GfcG GfgA fAfU fCfa fugu aucG ccag fcAf agaL augg 96 csas g AD- A- gsas 1425 A- usUf 1515 67580.1 135343.1 uugu 135344.1 scac CfcA CfaG fGfA fUfc fgac ucuG uggu fgAf gauL caau 96 csgs c AD- A- usgs 1426 A- usAf 1516 67573.1 135329.1 guga 135330.1 sucu CfaU GfgA fGfG fAfg fcuu ccaU ccag fgUf auaL cacc 96 asgs u AD- A- cscs 1427 A- usAf 1517 67600.1 135387.1 agau 135388.1 scau AfuG CfgU fCfC fCfg fcga ggcA cgau fuAf guaL ucug 96 gsas a AD- A- gsus 1428 A- usAf 1518 67603.1 135393.1 gguu 135394.1 sggu GfcA CfaC fGfU fCfc fggg acuG ugac fcAf cuaL acca 96 csas g AD- A- asgs 1429 A- usCf 1519 67598.1 135383.1 gucc 135384.1 suca CfaA CfuG fAfU fGfc fgcc auuU agug fgGf agaL gacc 96 usgs g AD- A- uscs 1430 A- usAf 1520 67564.1 135311.1 acuu 135312.1 sgac GfaG UfcG fGfA fCfc fggc uccU gagu fcAf cuaL agug 96 ascs u AD- A- asgs 1431 A- usCf 1521 67574.1 135331.1 ucua 135332.1 suga GfcA AfaG fGfA fAfa fuuc ucuG uuuc fcUf agaL agac 96 uscs g AD- A- asus 1432 A- usUf 1522 67590.1 135365.1 ucuu 135366.1 suua ticA GfcA fGfA fCfc fggu ucuG gcua faAf aaaL agaa 96 uscs u AD- A- usus 1433 A- asCf 1523 67572.1 135327.1 cuuu 135328.1 suuu CfaG AfgC fAfG fAfc fgug cucU cuaa fgAf aguL aaga 96 asus c AD- A- gsus 1434 A- asAf 1524 67582.1 135349.1 gcua 135350.1 saga AfaG UfgG fUfU fGfa fucc aacU cauc fuUf uuuL agca 96 cscs u AD- A- uscs 1435 A- usGf 1525 67607.1 135401.1 ccau 135402.1 suag CfuU CfuG fUfG fCfa fugc caaA agcu fgAf acaL uggg 96 asas a AD- A- csus 1436 A- usAf 1526 67571.1 135325.1 gccu 135326.1 succ GfuG UfcC fAfC fAfc fgug gucA gagg fcAf auaL ggca 96 gsgs g AD- A- usgs 1437 A- usCf 1527 67599.1 135385.1 ugac 135386.1 sugg GfuG GfaU fGfA fCfc fgga uccA uccc fcGf agaL ucac 96 asgs g AD- A- uscs 1438 A- asUf 1528 67554.1 135291.1 ugag 135292.1 saaa CfuG AfcC fAfG fAfa fuug cucA guuu fgCf uauL ucag 96 asgs g AD- A- asgs 1439 A- usAf 1529 67549.1 135281.1 uugg 135282.1 sgcu UfuU UfuU fUfA fCfa fuga uaaA aaag faCf cuaL caac 96 uscs a AD- A- usus 1440 A- usCf 1530 67567.1 135317.1 gguu 135318.1 suag UfuA CfuU fUfG fUfu faaa cauA agcu faAf agaL acca 96 ascs u AD- A- gsgs 1441 A- usUf 1531 67558.1 135299.1 uuuu 135300.1 sccu AfuG AfgC fAfA fUfu faag uucA cuag fuAf gauL aaac 96 csas a AD- A- gsus 1442 A- usUf 1532 67569.1 135321.1 uuua 135322.1 succ UfgA UfaG fAfA fCfu fagc uuuC uagg faUf aaaL aaaa 96 cscs a AD- A- usus 1443 A- usCf 1533 67548.1 135279.1 uuau 135280.1 suuc GfaA CfuA fAfA fGfc fgcu uuuU agga fcAf agaL uaaa 96 ascs c AD- A- usas 1444 A- usUf 1534 67566.1 135315.1 ugaa 135316.1 sugc AfaG UfuC fCfU fCfu fagg agcU aagc fuUf aauL ucau 96 asas a AD- A- csgs 1445 A- usCf 1535 67613.1 135413.1 uuaa 135414.1 scca UfuC AfcC fAfG fAfg fcug cugA guug faUf ggaL uaac 96 gscs a AD- A- gsus 1446 A- usUf 1536 67610.1 135407.1 uaau 135408.1 sccc ticA AfaC fGfC fCfa fugg gcuG uugg faAf gauL uuaa 96 csgs c AD- A- asgs 1447 A- usGf 1537 67556.1 135295.1 cugg 135296.1 sugu UfuG CfaU fGfG fUfu faaa cccA ugac faCf acaL cagc 96 usgs a AD- A- cscs 1448 A- asAf 1538 67581.1 135345.1 uauu 135346.1 scag AfaU UfcU fGfG fGfa fuca ccaU gacu fuAf guuL auag 96 gsgs c AD- A- csus 1449 A- usAf 1539 67560.1 135303.1 auua 135304.1 saca AfuG GfuC fGfU fUfg fcag accA acug fuUf uuaL aaua 96 gsgs g AD- A- gscs 1450 A- usAf 1540 67596.1 135379.1 uggc 135380.1 saga CfcA UfcA fUfG fCfa fugu cauG gauc fgGf uuaL ccag 96 cscs u AD- A- usgs 1451 A- usAf 1541 67557.1 135297.1 gccc 135298.1 scaa AfuG GfaU fUfG fCfa fuga cacA ucuu fuGf guaL ggcc 96 asgs c AD- A- cscs 1452 A- usUf 1542 67584.1 135353.1 uaac 135354.1 saaa UfaA CfaU fAfA fUfa fuaa uuuU uguu faGf uaaL uuag 96 gsus g AD- A- usus 1453 A- asAf 1543 67575.1 135333.1 accu 135334.1 suac GfuU AfaA fGfA fAfu fauu ucaA uugu fcAf auuL ggua 96 ascs a AD- A- ascs 1454 A- asUf 1544 67605.1 135397.1 cugu 135398.1 saau UfgA AfcA fAfU fAfa fuuu auuC guau faAf uauL cagg 96 usas a AD- A- usgs 1455 A- usUf 1545 67562.1 135307.1 uauu 135308.1 scac AfuG UfgA fUfG fUfu faau cacA cagu fuAf gaaL auac 96 asas a AD- A- usas 1456 A- asAf 1546 67606.1 135399.1 ugug 135400.1 scau AfaU CfuC fCfA fAfc fgug ugaU agau fuCf guuL acau 96 asas u AD- A- gsas 1457 A- asAf 1547 67604.1 135395.1 uguu 135396.1 sggc AfgU UfuA fAfG fUfu faau cuaC aagc fuAf cuuL acau 96 csus c AD- A- asus 1458 A- usAf 1548 67565.1 135313.1 guua 135314.1 sagg GfuA CfuU fGfA fAfu faua ucuA agcc fcUf uuaL aaca 96 uscs u AD- A- usas 1459 A- usCf 1549 67529.1 135241.1 uaau 135242.1 scau GfgA GfaG fGfA fGfa fucc ucuC ucau fcAf ggaL uuau 96 ascs g AD- A- gsus 1460 A- usUf 1550 67533.1 135249.1 gucu 135250.1 sugg GfaG AfaU fUfU fGfg fcca aacU uucc fcAf aaaL gaca 96 cscs a AD- A- asgs 1461 A- usAf 1551 67537.1 135257.1 ucgu 135258.1 scac GfgA CfaG fUfG fGfg fccc cauC uggu fcAf guaL cgac 96 usus c AD- A- usgs 1462 A- usUf 1552 67546.1 135275.1 cuau 135276.1 scca CfaA GfgU fGfG fAfc fgua ccuU ccug fgAf gauL uagc 96 ascs a AD- A- uscs 1463 A- usAf 1553 67547.1 135277.1 ccag 135278.1 suuc GfuU GfgG fUfG fCfa fugc caaA ccga fcCf auaL uggg 96 asus g AD- A- cscs 1464 A- usUf 1554 67543.1 135269.1 aggu 135270.1 scau UfuG UfcG fUfG fGfg fccc cacA gaau faAf gaaL ccug 96 gsgs a AD- A- usgs 1465 A- usAf 1555 67541.1 135265.1 gagc 135266.1 sucu AfaC AfgA fAfG fCfa fugu cugU cuag fuGf auaL cucc 96 asgs a AD- A- csus 1466 A- usUf 1556 67535.1 135253.1 uuug 135254.1 succ GfaG UfaG fGfC fCfu fagc gccU uagg fcCf aauL aaaa 96 gsus a AD- A- asas 1467 A- usAf 1557 67530.1 135243.1 gaca 135244.1 saac AfuG AfcC fAfU fAfa fuug aucA gugu fuUf uuaL gucu 96 usus g AD- A- gsas 1468 A- usCf 1558 67542.1 135267.1 caau 135268.1 suaa GfaU AfcA fUfU fCfc fggu aaaU guuu fcAf agaL uugu 96 csus u AD- A- csas 1469 A- usUf 1559 67528.1 135239.1 auga 135240.1 sucu UfuU AfaA fGfG fCfa fugu ccaA uuag faUf aaaL cauu 96 gsus c AD- A- usgs 1470 A- asAf 1560 67527.1 135237.1 ccag 135238.1 sagu AfuA AfaU fAfC fAfa fuua guuA uuac fuCf uuuL uggc 96 asgs g AD- A- ascs 1471 A- asUf 1561 67544.1 135271.1 accu 135272.1 suag UfuG UfaA fGfC fGfa fucu gccA uacu faAf aauL ggug 96 uscs c AD- A- csus 1472 A- usAf 1562 67532.1 135247.1 ggcu 135248.1 suac CfcA AfaA fAfA fGfa fucu uuuG uugu fgAf auaL gcca 96 gsus g AD- A- usgs 1473 A- usUf 1563 67534.1 135251.1 gcuc 135252.1 saua CfaA CfaA fAfU fAfg fcuu auuU ugua fgGf uaaL agcc 96 asgs u AD- A- cscs 1474 A- usAf 1564 67538.1 135259.1 aaau 135260.1 suga CfuU CfuA fUfG fUfa fuau caaA aguc fgAf auaL uuug 96 gsas g AD- A- asgs 1475 A- usAf 1565 67545.1 135273.1 agac 135274.1 sgcc AfaA UfaG fGfU fAfc fguc acuU uagg fuGf cuaL ucuc 96 usas g AD- A- asas 1476 A- usUf 1566 67539.1 135261.1 gugu 135262.1 scug CfuA UfgU fGfG fAfg fcua ccuA caca fgAf gaaL cacu 96 usus g AD- A- asgs 1477 A- usAf 1567 67540.1 135263.1 aaac 135264.1 saag UfuC CfaA fUfG fGfg fccu cagA ugcu faGf uuaL uuuc 96 usas c AD- A- gsas 1478 A- usGf 1568 67531.1 135245.1 agga 135246.1 sugu UfuG AfuC fAfA fCfa fugg uucA auac faUf acaL ccuu 96 csus g AD- A- gsgs 1479 A- usUf 1569 67536.1 135255.1 auug 135256.1 sugg AfaU UfgU fGfG fAfu faua ccaU cacc fuCf aaaL aauc 96 csus u

TABLE 9 PNPLA3 mRNA Target Sequences of Modifed PNPLA3 Agents in Table 8 Anti- mRNA Sense- ssense target SEQ Duplex Oligo Oligo se- ID Name Name Name quence NO AD- A- A- GCGGC 1570 67524.1 135231.1 135232.1 UUCCU GGGCU UCUAC CAC AD- A- A- UCUUG 1571 67611.1 135409.1 135410.1 UGCGG AAGGC CAGGA GUC AD- A- A- GGAAG 1572 67601.1 135389.1 135390.1 GCCAG GAGUC GGAAC AUU AD- A- A- GAAGG 1573 67579.1 135341.1 135342.1 CCAGG AGUCG GAACA UUG AD- A- A- AAAAC 1574 67588.1 135361.1 135362.1 GUUCU GGUGU CUGAC UUU AD- A- A- GUCUG 1575 67602.1 135391.1 135392.1 ACUUU CGGUC CAAAG ACG AD- A- A- UUUCG 1576 67570.1 135323.1 135324.1 GUCCA AAGAC GAAGU CGU AD- A- A- UUCGG 1577 67553.1 135289.1 135290.1 UCCAA AGACG AAGUC GUG AD- A- A- AAGAC 1578 67612.1 135411.1 135412.1 GAAGU CGUGG AUGCC UUG AD- A- A- GCCUU 1579 67525.1 135233.1 135234.1 GGUAU GUUCC UGCUU CAU AD- A- A- GUGGC 1580 67526.1 135235.1 135236.1 CUUAU CCCUC CUUCC UUC AD- A- A- GGAGG 1581 67592.1 135371.1 135372.1 AGUGA GUGAC AACGU ACC AD- A- A- GAGUG 1582 67578.1 135339.1 135340.1 AGUGA CAACG UACCC UUC AD- A- A- AUUGA 1583 67555.1 135293.1 135294.1 UGCCA AAACA ACCAU CAC AD- A- A- UACGA 1584 67577.1 135337.1 135338.1 CAUCU GCCCU AAAGU CAA AD- A- A- CCACG 1585 67594.1 135375.1 135376.1 AACUU UCUUC AUGUG GAC AD- A- A- CUGCA 1586 67568.1 135319.1 135320.1 CAGGG AACCU CUACC UUC AD- A- A- GGUGC 1587 67550.1 135283.1 135284.1 UGGGA GAGAU AUGCC UUC AD- A- A- GCUGG 1588 67586.1 135357.1 135358.1 GAGAG AUAUG CCUUC GAG AD- A- A- GGAGA 1589 67576.1 135335.1 135336.1 GAUAU GCCUU CGAGG AUA AD- A- A- AGAUA 1590 67563.1 135309.1 135310.1 UGCCU UCGAG GAUAU UUG AD- A- A- UAUGC 1591 67552.1 135287.1 135288.1 CUUCG AGGAU AUUUG GAU AD- A- A- GCAUU 1592 67608.1 135403.1 135404.1 CAGGU UCUUG GAAGA GAA AD- A- A- GUCAU 1593 67593.1 135373.1 135374.1 CCUCA GAAGG GAUGG AUC AD- A- A- AUCCU 1594 67609.1 135405.1 135406.1 GCCCU GGGAU GAGAG CAU AD- A- A- AAGAC 1595 67597.1 135381.1 135382.1 AAAGG UGGAU ACAUG AGC AD- A- A- ACAAA 1596 67587.1 135359.1 135360.1 GGUGG AUACA UGAGC AAG AD- A- A- AGGUG 1597 67559.1 135301.1 135302.1 GAUAC AUGAG CAAGA UUU AD- A- A- GGUGG 1598 67561.1 135305.1 135306.1 AUACA UGAGC AAGAU UUG AD- A- A- GGAUA 1599 67551.1 135285.1 135286.1 CAUGA GCAAG AUUUG CAA AD- A- A- UGAGC 1600 67591.1 135369.1 135370.1 AAGAU UUGCA ACUUG CUA AD- A- A- AGCAA 1601 67583.1 135351.1 135352.1 GAUUU GCAAC UUGCU ACC AD- A- A- UUUGC 1602 67585.1 135355.1 135356.1 AACUU GCUAC CCAUU AGG AD- A- A- GCAAC 1603 67589.1 135363.1 135364.1 UUGCU ACCCA UUAGG AUA AD- A- A- CUGCC 1604 67595.1 135377.1 135378.1 AUUGC GAUUG UCCAG AGA AD- A- A- GCGAU 1605 67580.1 135343.1 135344.1 UGUCC AGAGA CUGGU GAC AD- A- A- ACUGG 1606 67573.1 135329.1 135330.1 UGACA UGGCU UCCAG AUA AD- A- A- UUCCA 1607 67600.1 135387.1 135388.1 GAUAU GCCCG ACGAU GUC AD- A- A- CUGUG 1608 67603.1 135393.1 135394.1 GUUGC AGUGG GUGAC CUC AD- A- A- CCAGG 1609 67598.1 135383.1 135384.1 UCCCA AAUGC CAGUG AGC AD- A- A- AGUCA 1610 67564.1 135311.1 135312.1 CUUGA GGAGG CGAGU CUA AD- A- A- CGAGU 1611 67574.1 135331.1 135332.1 CUAGC AGAUU CUUUC AGA AD- A- A- AGAUU 1612 67590.1 135365.1 135366.1 CUUUC AGAGG UGCUA AAG AD- A- A- GAUUC 1613 67572.1 135327.1 135328.1 UUUCA GAGGU GCUAA AGU AD- A- A- AGGUG 1614 67582.1 135349.1 135350.1 CUAAA GUUUC CCAUC UUU AD- A- A- UUUCC 1615 67607.1 135401.1 135402.1 CAUCU UUGUG CAGCU ACC AD- A- A- CCCUG 1616 67571.1 135325.1 135326.1 CCUGU GACGU GGAGG AUC AD- A- A- CCUGU 1617 67599.1 135385.1 135386.1 GACGU GGAGG AUCCC AGC AD- A- A- CCUCU 1618 67554.1 135291.1 135292.1 GAGCU GAGUU GGUUU UAU AD- A- A- UGAGU 1619 67549.1 135281.1 135282.1 UGGUU UUAUG AAAAG CUA AD- A- A- AGUUG 1620 67567.1 135317.1 135318.1 GUUUU AUGAA AAGCU AGG AD- A- A- UUGGU 1621 67558.1 135299.1 135300.1 UUUAU GAAAA GCUAG GAA AD- A- A- UGGUU 1622 67569.1 135321.1 135322.1 UUAUG AAAAG CUAGG AAG AD- A- A- GGUUU 1623 67548.1 135279.1 135280.1 UAUGA AAAGC UAGGA AGC AD- A- A- UUUAU 1624 67566.1 135315.1 135316.1 GAAAA GCUAG GAAGC AAC AD- A- A- UGCGU 1625 67613.1 135413.1 135414.1 UAAUU CAGCU GGUUG GGA AD- A- A- GCGUU 1626 67610.1 135407.1 135408.1 AAUUC AGCUG GUUGG GAA AD- A- A- UCAGC 1627 67556.1 135295.1 135296.1 UGGUU GGGAA AUGAC ACC AD- A- A- GCCCU 1628 67581.1 135345.1 135346.1 AUUAA UGGUC AGACU GUU AD- A- A- CCCUA 1629 67560.1 135303.1 135304.1 UUAAU GGUCA GACUG UUC AD- A- A- AGGCU 1630 67596.1 135379.1 135380.1 GGCCC AUGUG UGAUC UUG AD- A- A- GCUGG 1631 67557.1 135297.1 135298.1 CCCAU GUGUG AUCUU GUG AD- A- A- CACCU 1632 67584.1 135353.1 135354.1 AACUA AAAUA AUGUU UAA AD- A- A- UGUUA 1633 67575.1 135333.1 135334.1 CCUGU UGAAU UUUGU AUU AD- A- A- UUACC 1634 67605.1 135397.1 135398.1 UGUUG AAUUU UGUAU UAU AD- A- A- UUUGU 1635 67562.1 135307.1 135308.1 AUUAU GUGAA UCAGU GAG AD- A- A- AUUAU 1636 67606.1 135399.1 135400.1 GUGAA UCAGU GAGAU GUU AD- A- A- GAGAU 1637 67604.1 135395.1 135396.1 GUUAG UAGAA UAAGC CUU AD- A- A- AGAUG 1638 67565.1 135313.1 135314.1 UUAGU AGAAU AAGCC UUA AD- A- A- CGUAU 1639 67529.1 135241.1 135242.1 AAUGG AGAUC CUCAU GGA AD- A- A- UGGUG 1640 67533.1 135249.1 135250.1 UCUGA GUUCC AUUCC AAA AD- A- A- GAAGU 1641 67537.1 135257.1 135258.1 CGUGG AUGCC CUGGU GUG AD- A- A- UGUGC 1642 67546.1 135275.1 135276.1 UAUCA AGGGU ACCUG GAC AD- A- A- CAUCC 1643 67547.1 135277.1 135278.1 CAGGU UUGUG CCCGA AUG AD- A- A- UCCCA 1644 67543.1 135269.1 135270.1 GGUUU GUGCC CGAAU GAC AD- A- A- UCUGG 1645 67541.1 135265.1 135266.1 AGCAA CAGUG UCUAG AUG AD- A- A- UACUU 1646 67535.1 135253.1 135254.1 UUGGA GGCAG CUAGG AAG AD- A- A- CAAAG 1647 67530.1 135243.1 135244.1 ACAAU GAUUU GGUGU UUA AD- A- A- AAGAC 1648 67542.1 135267.1 135268.1 AAUGA UUUGG UGUUU AGA AD- A- A- GACAA 1649 67528.1 135239.1 135240.1 UGAUU UGGUG UUUAG AAA AD- A- A- CCUGC 1650 67527.1 135237.1 135238.1 CAGAU AACUU AUUAC UUU AD- A- A- GGACA 1651 67544.1 135271.1 135272.1 CCUUU GGCUC UUACU AAU AD- A- A- CACUG 1652 67532.1 135247.1 135248.1 GCUCC AAAUC UUUGU AUA AD- A- A- ACUGG 1653 67534.1 135251.1 135252.1 CUCCA AAUCU UUGUA UAG AD- A- A- CUCCA 1654 67538.1 135259.1 135260.1 AAUCU UUGUA UAGUC AUC AD- A- A- CUAGA 1655 67545.1 135273.1 135274.1 GACAA AGUGU CUAGG CUA AD- A- A- CAAAG 1656 67539.1 135261.1 135262.1 UGUCU AGGCU ACACA GAA AD- A- A- GUAGA 1657 67540.1 135263.1 135264.1 AACUU CUGCC UUGCU UUG AD- A- A- CAGAA 1658 67531.1 135245.1 135246.1 GGAUU GAAUG GAUAC ACC AD- A- A- AAGGA 1659 67536.1 135255.1 135256.1 UUGAA UGGAU ACACC AAA

TABLE 10 Hep3B PNPLA3 endogenous in vitro 10 nM and 0.1 nM single dose screen Duplex 10 10 0.1 0.1 Name nM_AVG nM_STDEV nM_AVG nM_STDEV AD-67524.1 85.34 6.47 90.33 14.17 AD-67611.1 111.68 23.20 95.28 9.27 AD-67601.1 86.74 11.59 86.84 15.67 AD-67579.1 89.60 12.00 58.82 15.51 AD-67588.1 57.73 16.50 64.15 8.44 AD-67602.1 66.35 12.49 86.76 8.21 AD-67570.1 72.72 8.21 85.53 5.27 AD-67553.1 67.90 6.88 84.25 11.40 AD-67612.1 62.03 12.34 51.52 5.24 AD-67525.1 42.50 9.07 65.77 14.98 AD-67526.1 45.14 9.69 58.35 4.26 AD-67592.1 55.32 9.06 58.01 6.50 AD-67578.1 51.16 8.74 53.17 15.71 AD-67555.1 92.88 23.69 66.11 10.08 AD-67577.1 53.93 9.32 55.41 5.82 AD-67594.1 79.39 12.41 78.57 7.33 AD-67568.1 43.12 7.69 65.24 11.56 AD-67550.1 62.65 16.23 87.64 22.99 AD-67586.1 57.51 11.23 66.30 21.67 AD-67576.1 62.33 9.41 66.43 17.91 AD-67563.1 56.23 17.97 69.60 6.43 AD-67552.1 55.69 5.10 103.09 5.25 AD-67608.1 51.30 15.89 53.54 16.44 AD-67593.1 52.04 9.82 69.34 7.89 AD-67609.1 90.41 32.12 73.63 16.54 AD-67597.1 78.98 19.93 90.94 16.10 AD-67587.1 81.37 16.51 70.07 28.64 AD-67559.1 71.11 9.40 96.14 12.25 AD-67561.1 50.85 14.84 56.18 15.19 AD-67551.1 37.30 6.63 53.00 4.52 AD-67591.1 70.98 19.00 93.65 11.21 AD-67583.1 65.57 7.72 80.60 14.05 AD-67585.1 53.90 14.18 52.77 10.67 AD-67589.1 43.29 5.45 54.29 4.43 AD-67595.1 83.09 44.03 88.45 13.90 AD-67580.1 88.42 14.74 74.18 8.01 AD-67573.1 60.57 4.91 71.22 17.26 AD-67600.1 70.88 0.97 65.57 10.49 AD-67603.1 100.97 25.43 86.68 16.12 AD-67598.1 55.25 6.91 79.47 10.06 AD-67564.1 65.67 14.01 60.23 4.86 AD-67574.1 63.24 16.91 68.91 19.35 AD-67590.1 70.11 7.76 68.94 18.75 AD-67572.1 86.54 6.37 95.11 36.91 AD-67582.1 57.31 14.76 52.76 8.24 AD-67607.1 59.03 14.94 59.28 10.58 AD-67571.1 99.63 15.80 89.53 6.64 AD-67599.1 94.78 19.21 87.91 7.53 AD-67554.1 36.53 8.09 56.06 5.32 AD-67549.1 56.20 20.65 56.90 10.27 AD-67567.1 57.81 4.61 67.97 17.13 AD-67558.1 57.17 10.26 60.10 11.12 AD-67569.1 66.43 25.81 58.49 14.52 AD-67548.1 52.14 8.72 75.41 15.44 AD-67566.1 54.88 11.91 51.93 11.84 AD-67613.1 83.78 26.96 79.37 8.59 AD-67610.1 78.50 18.94 80.88 11.97 AD-67556.1 87.08 5.39 87.94 8.28 AD-67581.1 52.21 11.55 84.89 7.12 AD-67560.1 51.65 4.09 67.85 6.59 AD-67596.1 82.71 20.80 76.57 11.58 AD-67557.1 56.15 8.28 90.70 5.11 AD-67584.1 42.16 6.42 38.63 13.85 AD-67575.1 42.62 11.19 54.35 9.20 AD-67605.1 43.75 11.62 59.95 7.68 AD-67562.1 73.26 11.12 72.58 11.11 AD-67606.1 86.42 38.80 75.45 12.67 AD-67604.1 64.47 6.80 72.33 10.76 AD-67565.1 49.43 3.37 54.34 12.25 AD-67529.1 96.11 23.73 104.54 5.56 AD-67533.1 91.29 27.25 102.72 10.83 AD-67537.1 96.12 30.20 90.92 17.55 AD-67546.1 117.18 35.85 90.75 10.80 AD-67547.1 109.66 23.27 110.07 17.90 AD-67543.1 106.67 27.98 103.10 22.41 AD-67541.1 112.89 34.51 105.50 18.29 AD-67535.1 95.95 17.30 111.96 8.37 AD-67530.1 86.64 13.15 89.64 10.56 AD-67542.1 108.30 12.22 111.03 18.93 AD-67528.1 86.06 15.40 100.52 11.52 AD-67527.1 94.22 9.43 103.95 8.31 AD-67544.1 95.63 16.01 94.25 5.66 AD-67532.1 96.24 10.13 114.20 14.38 AD-67534.1 104.27 20.55 101.24 14.18 AD-67538.1 108.29 29.79 99.37 10.01 AD-67545.1 110.68 11.06 143.56 45.88 AD-67539.1 106.92 43.45 107.56 15.77 AD-67540.1 104.01 18.83 105.58 12.67 AD-67531.1 117.06 37.65 102.32 27.15 AD-67536.1 104.51 7.42 110.11 14.23

TABLE 11 Cynomolgus monkey PNPLA3 endogenous in vitro 10 nM and 0.1 nM single dose screen Duplex 10 10 0.1 0.1 Name nM_AVG nM_STDEV nM_AVG nM_STDEV AD-67524.1 64.22 12.50 98.57 57.56 AD-67611.1 201.95 55.02 147.71 34.65 AD-67601.1 106.76 23.66 104.01 20.80 AD-67579.1 69.15 24.02 39.69 7.49 AD-67588.1 34.18 13.04 58.34 19.48 AD-67602.1 64.07 21.95 114.16 40.22 AD-67570.1 45.66 21.83 92.73 22.46 AD-67553.1 61.54 20.51 78.87 33.03 AD-67612.1 49.05 10.63 68.98 21.48 AD-67525.1 58.61 6.56 83.50 29.86 AD-67526.1 48.75 19.00 81.70 44.79 AD-67592.1 54.34 23.45 107.45 52.70 AD-67578.1 54.22 18.19 62.05 18.44 AD-67555.1 83.45 13.63 96.21 32.86 AD-67577.1 41.40 13.97 50.80 20.40 AD-67594.1 71.17 26.23 90.30 12.23 AD-67568.1 28.74 8.05 56.57 12.90 AD-67550.1 67.27 14.09 102.11 22.04 AD-67586.1 44.83 10.13 52.06 1.96 AD-67576.1 61.04 36.58 78.16 7.18 AD-67563.1 85.83 27.55 88.34 7.26 AD-67552.1 70.65 36.42 112.67 14.77 AD-67608.1 65.16 37.26 90.87 21.05 AD-67593.1 72.95 19.92 108.58 27.09 AD-67609.1 83.80 52.06 113.25 23.43 AD-67597.1 57.86 7.16 101.52 29.68 AD-67587.1 71.36 33.38 83.46 28.71 AD-67559.1 38.13 5.57 85.54 20.52 AD-67561.1 49.61 17.03 75.51 35.59 AD-67551.1 24.74 13.01 57.84 19.55 AD-67591.1 65.58 11.64 70.61 18.06 AD-67583.1 35.16 12.01 56.71 13.29 AD-67585.1 51.64 38.68 91.09 23.58 AD-67589.1 30.43 8.50 55.59 15.49 AD-67595.1 64.53 12.69 108.07 61.59 AD-67580.1 52.22 14.63 59.80 19.21 AD-67573.1 47.55 19.02 69.12 8.02 AD-67600.1 55.58 11.69 92.41 26.52 AD-67603.1 119.04 50.54 152.95 37.00 AD-67598.1 51.72 17.51 84.34 25.38 AD-67564.1 58.62 27.17 77.33 37.58 AD-67574.1 33.51 14.78 45.90 17.45 AD-67590.1 40.45 9.84 56.63 12.25 AD-67572.1 47.06 14.49 77.89 27.67 AD-67582.1 27.10 5.89 49.41 18.11 AD-67607.1 43.61 8.27 72.35 13.09 AD-67571.1 109.27 56.41 69.47 23.50 AD-67599.1 83.03 58.74 83.94 15.01 AD-67554.1 19.86 10.03 85.24 13.88 AD-67549.1 38.63 14.53 94.17 23.93 AD-67567.1 31.60 11.57 71.66 11.57 AD-67558.1 39.31 19.51 67.91 23.15 AD-67569.1 35.42 13.52 37.45 9.62 AD-67548.1 84.14 21.27 83.38 26.65 AD-67566.1 26.85 5.90 47.24 9.79 AD-67613.1 90.32 43.98 110.21 22.37 AD-67610.1 76.90 29.15 116.95 25.59 AD-67556.1 99.65 38.94 78.32 27.42 AD-67581.1 31.34 9.79 69.04 11.53 AD-67560.1 25.86 10.00 49.96 14.82 AD-67596.1 70.39 22.12 83.45 32.21 AD-67557.1 30.36 3.67 77.23 33.61 AD-67584.1 30.34 10.44 35.30 9.75 AD-67575.1 29.04 9.17 48.86 8.65 AD-67605.1 62.92 35.30 97.67 46.22 AD-67562.1 149.14 76.05 137.22 31.54 AD-67606.1 53.08 12.65 76.76 17.13 AD-67604.1 45.22 6.49 90.48 27.49 AD-67565.1 58.35 24.21 60.94 28.29 AD-67529.1 158.59 45.47 150.25 53.50 AD-67533.1 142.31 43.60 146.81 39.93 AD-67537.1 141.43 43.53 173.26 50.75 AD-67546.1 176.38 88.77 147.25 35.28 AD-67547.1 160.76 104.70 125.22 35.52 AD-67543.1 117.94 26.94 178.90 44.99 AD-67541.1 171.26 52.40 148.66 41.86 AD-67535.1 117.80 12.65 154.87 34.75 AD-67530.1 130.28 46.60 124.85 37.32 AD-67542.1 130.98 44.83 158.70 46.06 AD-67528.1 131.06 56.44 149.25 40.56 AD-67527.1 128.94 29.24 154.29 24.13 AD-67544.1 122.80 57.17 155.85 21.25 AD-67532.1 73.68 20.38 130.31 58.83 AD-67534.1 173.61 86.25 174.54 61.47 AD-67538.1 153.55 53.00 170.55 45.06 AD-67545.1 139.49 20.95 128.18 37.75 AD-67539.1 258.50 123.06 144.40 39.80 AD-67540.1 139.83 54.43 134.33 34.88 AD-67531.1 131.80 41.72 155.34 63.49 AD-67536.1 143.28 42.58 150.88 41.76

TABLE 12 Cynomolgus monkey PNPLA3 endogenous in vitro dose response screen Duplex Name IC₅₀ (nM) AD-67525.1 0.003 AD-67526.1 0.005 AD-67551.1 0.298 AD-67554.1 0.003 AD-67560.1 0.034 AD-67568.1 0.049 AD-67575.1 0.317 AD-67577.1 0.001 AD-67578.1 0.001 AD-67581.1 0.081 AD-67582.1 0.058 AD-67584.1 0.001 AD-67585.1 0.038 AD-67592.1 0.216 AD-67605.1 0.123 AD-67612.1 1.381

TABLE 13 Mouse PNPLA3 endogenous in vitro 10 nM and 0.1 nM single dose screen Duplex 10 10 0.1 0.1 Name nM_AVG nM_STDEV nM_AVG nM_STDEV AD-67524.1 59.20 19.07 172.14 102.64 AD-67611.1 101.53 39.04 166.84 16.96 AD-67601.1 106.69 30.70 133.40 10.57 AD-67579.1 93.33 23.59 111.59 17.65 AD-67588.1 87.03 12.30 114.60 9.93 AD-67602.1 99.22 9.70 127.47 8.95 AD-67570.1 19.23 14.62 74.87 29.73 AD-67553.1 15.43 7.94 60.49 46.00 AD-67612.1 28.43 11.87 91.40 64.47 AD-67525.1 39.75 26.63 140.88 39.79 AD-67526.1 16.16 5.74 97.66 43.58 AD-67592.1 25.04 16.02 117.35 19.59 AD-67578.1 27.07 18.71 138.94 57.91 AD-67555.1 43.71 36.83 148.89 53.90 AD-67577.1 35.95 24.04 106.43 62.19 AD-67594.1 118.99 24.43 118.98 22.56 AD-67568.1 22.01 15.09 104.04 28.73 AD-67550.1 128.32 33.32 153.22 21.77 AD-67586.1 74.59 3.44 106.75 20.87 AD-67576.1 79.48 5.82 129.09 31.89 AD-67563.1 141.90 59.30 132.24 34.38 AD-67552.1 143.18 49.65 124.20 18.26 AD-67608.1 154.76 66.58 190.93 42.10 AD-67593.1 112.21 46.12 116.10 14.11 AD-67609.1 164.89 46.54 171.23 27.85 AD-67597.1 145.67 37.55 143.83 34.86 AD-67587.1 102.09 20.42 106.63 18.56 AD-67559.1 126.57 13.72 137.00 24.64 AD-67561.1 121.82 26.66 151.62 35.30 AD-67551.1 152.46 60.75 133.95 21.61 AD-67591.1 166.93 65.07 145.36 28.46 AD-67583.1 127.60 32.09 142.44 42.81 AD-67585.1 99.84 23.69 148.35 48.23 AD-67589.1 106.32 18.73 156.26 53.14 AD-67595.1 105.40 18.78 123.24 28.02 AD-67580.1 105.49 27.33 127.82 10.97 AD-67573.1 17.45 7.76 126.15 41.24 AD-67600.1 86.36 21.17 126.08 20.47 AD-67603.1 104.95 35.50 142.30 15.52 AD-67598.1 95.85 23.74 172.20 35.33 AD-67564.1 109.00 17.65 121.28 23.44 AD-67574.1 86.31 11.33 131.22 27.38 AD-67590.1 136.21 58.52 123.09 14.38 AD-67572.1 139.23 24.55 115.97 17.05 AD-67582.1 126.01 33.04 165.25 40.55 AD-67607.1 94.42 35.45 121.14 19.57 AD-67571.1 112.27 26.92 120.03 21.17 AD-67599.1 171.97 24.42 113.09 20.69 AD-67554.1 125.76 26.14 118.60 35.12 AD-67549.1 119.56 65.06 150.20 16.69 AD-67567.1 133.44 93.04 144.51 44.20 AD-67558.1 158.66 58.69 115.42 22.09 AD-67569.1 123.35 42.23 150.79 30.96 AD-67548.1 130.24 31.18 126.72 29.14 AD-67566.1 97.88 15.18 161.34 45.64 AD-67613.1 133.15 53.50 164.06 35.86 AD-67610.1 125.68 41.94 123.89 17.82 AD-67556.1 129.25 45.87 156.50 34.41 AD-67581.1 81.75 13.75 127.10 26.78 AD-67560.1 119.69 56.51 127.65 12.06 AD-67596.1 104.08 30.46 128.33 24.04 AD-67557.1 78.91 9.50 127.50 9.39 AD-67584.1 131.87 19.40 128.29 20.96 AD-67575.1 124.30 43.53 151.26 44.05 AD-67605.1 122.92 37.28 120.37 16.17 AD-67562.1 124.35 35.73 109.59 17.85 AD-67606.1 160.77 45.92 152.73 33.64 AD-67604.1 111.98 13.56 167.29 31.28 AD-67565.1 135.81 13.80 120.59 16.82 AD-67529.1 38.14 15.75 121.25 64.05 AD-67533.1 12.73 5.26 16.84 7.89 AD-67537.1 86.70 19.46 92.22 10.90 AD-67546.1 51.08 19.80 93.17 14.25 AD-67547.1 23.08 14.10 64.05 22.49 AD-67543.1 79.50 28.07 111.48 34.28 AD-67541.1 18.70 11.01 55.21 13.26 AD-67535.1 36.56 12.86 93.32 28.28 AD-67530.1 33.36 9.33 41.89 15.37 AD-67542.1 23.31 7.40 84.60 16.44 AD-67528.1 17.24 5.43 27.71 5.90 AD-67527.1 19.79 1.80 37.50 17.14 AD-67544.1 11.14 3.90 24.01 9.11 AD-67532.1 19.67 6.21 45.02 22.68 AD-67534.1 15.07 3.74 42.41 21.95 AD-67538.1 10.11 1.51 46.11 9.50 AD-67545.1 24.38 5.53 69.48 8.00 AD-67539.1 27.60 4.13 80.14 19.65 AD-67540.1 19.25 4.32 64.45 17.14 AD-67531.1 33.63 15.63 50.55 16.20 AD-67536.1 10.87 5.44 39.87 14.64

TABLE 14 Mouse PNPLA3 endogenous in vitro dose response screen Duplex Name IC₅₀ (nM) AD-67525.1 n/a AD-67526.1 n/a AD-67527.1 2.309 AD-67528.1 0.673 AD-67530.1 0.921 AD-67531.1 0.581 AD-67532.1 1.425 AD-67533.1 0.567 AD-67534.1 4.128 AD-67536.1 2.288 AD-67538.1 0.538 AD-67544.1 0.608 AD-67577.1 n/a AD-67578.1 n/a

TABLE 15 Human PNPLA3 Dual-Glo ® in vitro 10 nM and 0.1 nM single dose screen Duplex 10 10 0.1 0.1 Name nM_AVG nM_STDEV nM_AVG nM_STDEV AD-67524.1 55.85 7.32 102.37 32.69 AD-67611.1 107.30 22.70 97.85 2.28 AD-67601.1 95.78 11.23 85.88 29.26 AD-67579.1 77.51 8.06 101.67 36.37 AD-67588.1 47.11 6.33 89.10 18.85 AD-67602.1 77.70 12.22 81.37 13.84 AD-67570.1 48.66 10.55 68.57 28.45 AD-67553.1 44.56 7.76 93.93 38.43 AD-67612.1 38.37 0.82 75.31 20.53 AD-67525.1 29.59 9.56 62.31 9.28 AD-67526.1 53.90 3.79 70.76 15.61 AD-67592.1 52.84 10.35 98.32 32.26 AD-67578.1 53.64 4.02 64.89 13.47 AD-67555.1 56.39 10.80 69.54 19.05 AD-67577.1 48.65 10.91 54.23 6.43 AD-67594.1 74.48 1.78 93.18 24.59 AD-67568.1 45.05 1.82 92.90 28.78 AD-67550.1 44.13 4.53 68.69 20.38 AD-67586.1 64.19 5.26 84.24 40.47 AD-67576.1 79.72 15.25 82.61 35.92 AD-67563.1 29.23 4.19 59.00 12.91 AD-67552.1 54.78 11.92 79.03 16.42 AD-67608.1 57.15 4.59 77.51 11.84 AD-67593.1 101.86 11.04 96.29 17.81 AD-67609.1 122.26 11.52 128.58 16.30 AD-67597.1 62.83 5.62 101.58 38.86 AD-67587.1 57.38 14.11 105.10 27.16 AD-67559.1 51.66 5.22 91.89 14.10 AD-67561.1 51.41 3.67 71.48 29.44 AD-67551.1 34.40 1.24 49.32 10.41 AD-67591.1 60.13 3.19 90.29 18.00 AD-67583.1 36.86 2.53 93.04 25.60 AD-67585.1 53.41 8.56 79.44 18.57 AD-67589.1 37.94 7.73 75.57 10.11 AD-67595.1 75.77 8.06 90.18 31.68 AD-67580.1 71.41 1.40 74.67 23.67 AD-67573.1 70.86 4.99 84.53 20.34 AD-67600.1 72.31 16.88 78.24 10.32 AD-67603.1 75.73 13.27 83.86 13.43 AD-67598.1 77.98 14.11 86.38 27.50 AD-67564.1 75.61 4.75 112.02 16.56 AD-67574.1 60.65 11.08 83.89 27.59 AD-67590.1 58.37 10.03 73.59 23.40 AD-67572.1 96.15 19.05 99.37 14.65 AD-67582.1 35.14 4.73 61.85 10.01 AD-67607.1 35.55 8.50 66.52 7.13 AD-67571.1 87.08 10.17 90.89 10.55 AD-67599.1 102.26 6.41 94.34 3.26 AD-67554.1 47.64 4.28 69.79 12.50 AD-67549.1 27.66 2.50 51.69 14.59 AD-67567.1 37.31 6.07 64.11 19.16 AD-67558.1 30.72 4.79 72.23 22.28 AD-67569.1 36.42 1.62 68.82 8.06 AD-67548.1 59.63 13.22 94.53 28.41 AD-67566.1 54.82 11.76 58.13 29.99 AD-67613.1 70.40 10.36 69.65 14.48 AD-67610.1 75.42 12.56 83.75 10.26 AD-67556.1 84.41 2.30 91.86 36.53 AD-67581.1 53.86 14.24 100.03 42.15 AD-67560.1 40.96 10.50 64.75 25.60 AD-67596.1 75.01 9.11 99.67 17.69 AD-67557.1 46.45 5.85 82.71 7.14 AD-67584.1 30.32 1.09 29.60 4.38 AD-67575.1 18.95 5.11 34.22 7.00 AD-67605.1 18.06 8.25 31.33 15.81 AD-67562.1 53.05 12.05 65.06 24.50 AD-67606.1 27.53 7.98 44.22 17.40 AD-67604.1 51.35 1.71 78.70 19.78 AD-67565.1 19.72 1.66 44.43 16.60

TABLE 16 Human PNPLA3 Dual-Glo  in vitro dose response screen Duplex Name IC₅₀ (nM) AD-67584.1 0.1149 AD-67605.1 0.0915 AD-67575.1 0.1616 AD-67606.1 0.5824 AD-67565.1 0.1988 AD-67551.1 0.6022 AD-67549.1 0.7905

Example 4. In Vivo Effect of Single Dose Administration of PNPLA3 iRNA Agent

Ob/ob mice strongly express PNPLA3 in the liver. Accordingly, Ob/ob mice (B6.Cg-Lepob/J) were administered a single subcutaneous dose of 0.3 mg/kg, 1.5 mg/kg, or 3.0 mg/kg, or PBS alone as a control, of AD-67525, AD-67526, AD-67528, AD-65731, AD67533, AD-67538, or AD-67544. The animals were sacrificed and the livers were excised 96 hours post-dose and the level of PNPLA3 mRNA was quantified by RT-qPCR.

As shown in FIG. 1, AD-65726 administered as a single 1.5. mg/kg dose, or AD-67533 administered as a single 3.0 mg/kg dose exhibited the most robust suppression of hepatic PNPLA3 of the agents and doses assayed.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 

1. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein said double stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3-5, 7, and
 8. 2. The double stranded RNAi agent of claim 1, wherein said double stranded RNAi agent comprises at least one modified nucleotide.
 3. The double stranded RNAi agent of claim 2, wherein the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic.
 4. The double stranded RNAi agent of claim 1, wherein the region of complementarity is at least 17 nucleotides in length; between 19 and 21 nucleotides in length; or 19 nucleotides in length.
 5. The double stranded RNAi agent of claim 1, wherein each strand is no more than 30 nucleotides in length.
 6. The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide; or at least 2 nucleotides.
 7. The double stranded RNAi agent of claim 1, further comprising a ligand.
 8. A double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (III): (III) sense: 5′ n_(p)-Na-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′- (Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.
 9. The double stranded RNAi agent of claim 8, wherein formula (III) is represented by a formula selected from the group consisting of formula (IIIa): (IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′;

formula (IIIb): (IIIb) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides; formula (IIIc): (IIIc) sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b)-Y′Y′Y′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides; and formula (IIId): (IIId) sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)- n_(q) 3′ antisense: 3′ n_(P′)-N_(a′)-X′X′X′-N_(b)-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a)-n_(q)-5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.
 10. The double stranded RNAi agent of claim 7 or 8, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
 11. The double stranded RNAi agent of claim 10, the ligand is


12. The double stranded RNAi agent of claim 1 or 8, wherein said double stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
 13. A cell containing the double stranded RNAi agent of claim
 1. 14. A pharmaceutical composition for inhibiting expression of a PNPLA3 gene comprising the double stranded RNAi agent of claim
 1. 15. A method of inhibiting PNPLA3 expression in a cell, the method comprising: (a) contacting the cell with the double stranded RNAi agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the cell.
 16. The method of claim 15, wherein said cell is within a subject.
 17. The method of claim 16, wherein the subject is a human.
 18. A method of treating a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of claim 1, thereby treating said subject.
 19. A method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a prophylactically effective amount of the double stranded RNAi agent of claim 1, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 expression.
 20. The method of claim 18 or 19, wherein the disorder is a PNPLA3-associated disease.
 21. The method of any one of claim 18 or 19, further comprising administering an anti-PNPLA3 antibody, or antigen-binding fragment thereof, to the subject. 